RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (siNA)

ABSTRACT

This invention relates to compounds, compositions, and methods useful for modulating interleukin and/or interleukin receptor gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of interleukin and/or interleukin receptor gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of interleukin and/or interleukin receptor genes.

This application is a continuation of U.S. patent application Ser. No.10/863,973, filed on Jun. 9, 2004 (now abandoned), which is acontinuation-in-part of International Patent Application No.PCT/US03/04566, filed Feb. 14, 2003, and parent U.S. patent applicationSer. No. 10/863,973 (now abandoned), is also a continuation-in-part ofInternational Patent Application No. PCT/US04/16390, filed May 24, 2004,which is a continuation-in-part of U.S. patent application Ser. No.10/826,966, filed Apr. 16, 2004 (now abandoned), which iscontinuation-in-part of U.S. patent application Ser. No. 10/757,803,filed Jan. 14, 2004, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/720,448, filed Nov. 24, 2003, which is acontinuation-in-part of U.S. patent application Ser. No. 10/693,059,filed Oct. 23, 2003, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/444,853, filed May 23, 2003, which is acontinuation-in-part of International Patent Application No.PCT/US03/05346, filed Feb. 20, 2003, and a continuation-in-part ofInternational Patent Application No. PCT/US03/05028, filed Feb. 20,2003, both of which claim the benefit of U.S. Provisional ApplicationNo. 60/358,580, filed Feb. 20, 2002 (now abandoned), U.S. ProvisionalApplication No. 60/363,124, filed Mar. 11, 2002 (now abandoned), U.S.Provisional Application No. 60/386,782, filed Jun. 6, 2002 (nowabandoned), U.S. Provisional Application No. 60/406,784, filed Aug. 29,2002 (now abandoned), U.S. Provisional Application No. 60/408,378, filedSep. 5, 2002 (now abandoned), U.S. Provisional Application No.60/409,293, filed Sep. 9, 2002 (now abandoned), and U.S. ProvisionalApplication No. 60/440,129, filed Jan. 15, 2003 (now abandoned). Theinstant application claims the benefit of all the listed applications,which are hereby incorporated by reference herein in their entireties,including the drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing17USCNT”,created on Sep. 3, 2008, which is 483,552 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of interleukin gene expressionand/or activity, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, and IL-27 genesand genes encoding interleukin receptors of IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25,IL-26, and IL-27 genes. The present invention is also directed tocompounds, compositions, and methods relating to traits, diseases andconditions that respond to the modulation of expression and/or activityof genes involved in interleukin gene expression pathways or othercellular processes that mediate the maintenance or development of suchtraits, diseases and conditions. Specifically, the invention relates tosmall nucleic acid molecules, such as short interfering nucleic acid(siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable ofmediating RNA interference (RNAi) against interleukin gene expression.Such small nucleic acid molecules are useful, for example, in providingcompositions for treatment or prevention of traits, diseases andconditions that can respond to modulation of interleukin gene expressionin a subject, such as inflammatory, respiratory, pulmonary, autoimmune,cardiovascular, neurodegenerative, and/or proliferative and cancerousdiseases, traits, or conditions.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized. This mechanism appearsto be different from other known mechanisms involving double-strandedRNA-specific ribonucleases, such as the interferon response that resultsfrom dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094;5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17,503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of an siRNAduplex is required for siRNA activity and that ATP is utilized tomaintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation ofdouble-stranded RNA-dependent protein kinase PKR, specifically 2′-aminoor 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Pachuck et al., International PCTPublication No. WO 00/63364, describe certain long (at least 200nucleotide) dsRNA constructs. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA expressionconstructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically-modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al., International PCT Publication No. WO 02/38805,describe certain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certainsingle-stranded siRNA constructs, including certain 5′-phosphorylatedsingle-stranded siRNAs that mediate RNA interference in HeLa cells.Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13,83-105, describe certain chemically and structurally modified siRNAmolecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certainchemically and structurally modified siRNA molecules. Woolf et al.,International PCT Publication Nos. WO 03/064626 and WO 03/064625describe certain chemically modified dsRNA constructs.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating interleukin and/or interleukin receptor gene expressionusing short interfering nucleic acid (siNA) molecules. This inventionalso relates to compounds, compositions, and methods useful formodulating the expression and activity of other genes involved inpathways of interleukin and/or interleukin receptor gene expressionand/or activity by RNA interference (RNAi) using small nucleic acidmolecules. In particular, the instant invention features small nucleicacid molecules, such as short interfering nucleic acid (siNA), shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),and short hairpin RNA (shRNA) molecules and methods used to modulate theexpression of interleukin and/or interleukin receptor genes.

An siNA of the invention can be unmodified or chemically-modified. AnsiNA of the instant invention can be chemically synthesized, expressedfrom a vector or enzymatically synthesized. The instant invention alsofeatures various chemically-modified synthetic short interfering nucleicacid (siNA) molecules capable of modulating interleukin and/orinterleukin receptor gene expression or activity in cells by RNAinterference (RNAi). The use of chemically-modified siNA improvesvarious properties of native siNA molecules through increased resistanceto nuclease degradation in vivo and/or through improved cellular uptake.Further, contrary to earlier published studies, siNA having multiplechemical modifications retains its RNAi activity. The siNA molecules ofthe instant invention provide useful reagents and methods for a varietyof therapeutic, diagnostic, target validation, genomic discovery,genetic engineering, and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules andmethods that independently or in combination modulate the expression ofinterleukin and/or interleukin receptor genes encoding proteins, such asproteins comprising interleukins (e.g., IL-1-IL-27) and interleukinreceptors (e.g., IL-IR-1L-27R), such as genes encoding sequencescomprising those sequences referred to by GenBank Accession Nos. shownin Table I, referred to herein generally as interleukin and/orinterleukin receptor. The description below of the various aspects andembodiments of the invention is provided with reference to exemplaryinterleukin and interleukin receptor genes referred to herein asinterleukin and/or interleukin receptor. However, the various aspectsand embodiments are also directed to other interleukin and/orinterleukin receptor genes, such as interleukin and/or interleukinreceptor homolog genes, transcript variants, and polymorphisms (e.g.,single nucleotide polymorphism, (SNPs)) associated with certaininterleukin and/or interleukin receptor genes, for example genesassociated with diseases, traits, or conditions described herein orotherwise known in the art. As such, the various aspects and embodimentsare also directed to other genes that are involved in interleukin and/orinterleukin receptor mediated pathways of signal transduction or geneexpression. These additional genes can be analyzed for target sitesusing the methods described for interleukin and/or interleukin receptorgenes herein. Thus, the modulation of other genes and the effects ofsuch modulation of the other genes can be performed, determined, andmeasured as described herein.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a interleukin and/or interleukin receptor gene, wherein said siNAmolecule comprises about 19 to about 21 base pairs.

In one embodiment, the invention features an siNA molecule thatdown-regulates expression of a interleukin and/or interleukin receptorgene, for example, wherein the interleukin and/or interleukin receptorgene comprises interleukin and/or interleukin receptor encodingsequence. In one embodiment, the invention features an siNA moleculethat down-regulates expression of a interleukin and/or interleukinreceptor gene, for example, wherein the interleukin and/or interleukinreceptor gene comprises interleukin and/or interleukin receptornon-coding sequence or regulatory elements involved in interleukinand/or interleukin receptor gene expression.

In one embodiment, an siNA of the invention is used to inhibit theexpression of interleukin and/or interleukin receptor genes or ainterleukin and/or interleukin receptor gene family, wherein the genesor gene family sequences share sequence homology. Such homologoussequences can be identified as is known in the art, for example usingsequence alignments. siNA molecules can be designed to target suchhomologous sequences, for example using perfectly complementarysequences or by incorporating non-canonical base pairs, for examplemismatches and/or wobble base pairs, that can provide additional targetsequences. In instances where mismatches are identified, non-canonicalbase pairs, for example mismatches and/or wobble bases, can be used togenerate siNA molecules that target both more than one gene sequences.In a non-limiting example, non-canonical base pairs such as UU and CCbase pairs are used to generate siNA molecules that are capable oftargeting sequences for differing interleukin and/or interleukinreceptor targets that share sequence homology (e.g., differinginterleukin genes or differing allelic variants thereof). As such, oneadvantage of using siNAs of the invention is that a single siNA can bedesigned to include nucleic acid sequence that is complementary to thenucleotide sequence that is conserved between the homologous genes. Inthis approach, a single siNA can be used to inhibit expression of morethan one interleukin and/or interleukin receptor gene instead of usingmore than one siNA molecule to target the different genes.

In one embodiment, the invention features an siNA molecule having RNAiactivity against interleukin and/or interleukin receptor RNA, whereinthe siNA molecule comprises a sequence complementary to any RNA havinginterleukin and/or interleukin receptor encoding sequence, such as thosesequences having GenBank Accession Nos. shown in Table I. In anotherembodiment, the invention features an siNA molecule having RNAi activityagainst interleukin and/or interleukin receptor RNA, wherein the siNAmolecule comprises a sequence complementary to an RNA having variantinterleukin and/or interleukin receptor encoding sequence, for exampleother mutant interleukin and/or interleukin receptor genes not shown inTable I but known in the art to be associated with diseases, traits, orconditions described herein or otherwise known in the art. Chemicalmodifications as shown in Tables III and IV or otherwise describedherein can be applied to any siNA construct of the invention. In anotherembodiment, an siNA molecule of the invention includes a nucleotidesequence that can interact with nucleotide sequence of a interleukinand/or interleukin receptor gene and thereby mediate silencing ofinterleukin and/or interleukin receptor gene expression, for example,wherein the siNA mediates regulation of interleukin and/or interleukinreceptor gene expression by cellular processes that modulate thechromatin structure or methylation patterns of the interleukin and/orinterleukin receptor gene and prevent transcription of the interleukinand/or interleukin receptor gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of interleukin and/or interleukinreceptor proteins arising from interleukin and/or interleukin receptorhaplotype polymorphisms that are associated with a disease or condition,(e.g., proliferative, inflammatory, autoimmune, respiratory, pulmonary,cardiovascular, neurodegenerative diseases). Analysis of interleukinand/or interleukin receptor genes, or interleukin and/or interleukinreceptor protein or RNA levels can be used to identify subjects withsuch polymorphisms or those subjects who are at risk of developingtraits, conditions, or diseases described herein. These subjects areamenable to treatment, for example, treatment with siNA molecules of theinvention and any other composition useful in treating diseases relatedto interleukin and/or interleukin receptor gene expression. As such,analysis of interleukin and/or interleukin receptor protein or RNAlevels can be used to determine treatment type and the course of therapyin treating a subject. Monitoring of interleukin and/or interleukinreceptor protein or RNA levels can be used to predict treatment outcomeand to determine the efficacy of compounds and compositions thatmodulate the level and/or activity of certain interleukin and/orinterleukin receptor proteins associated with a trait, condition, ordisease.

In one embodiment of the invention an siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a interleukinand/or interleukin receptor protein. The siNA further comprises a sensestrand, wherein said sense strand comprises a nucleotide sequence of ainterleukin and/or interleukin receptor gene or a portion thereof.

In another embodiment, the invention features an siNA moleculecomprising a nucleotide sequence in the antisense region of the siNAmolecule that is complementary to a nucleotide sequence or portion ofsequence of a interleukin and/or interleukin receptor gene. In anotherembodiment, the invention features an siNA molecule comprising a region,for example, the antisense region of the siNA construct, complementaryto a sequence comprising a interleukin and/or interleukin receptor genesequence or a portion thereof.

In one embodiment, the antisense region of interleukin and/orinterleukin receptor siNA constructs comprises a sequence complementaryto sequence having any of SEQ ID NOs. 1-81, 163-213, 265-464, 665-735,807-1029, or 1253-1260. In one embodiment, the antisense region ofinterleukin and/or interleukin receptor constructs comprises sequencehaving any of SEQ ID NOs. 82-162, 214-264, 465-664, 736-806, 1030-1252,1319-1326, 1335-1342, 1351-1358, 1367-1374, 1383-1406, 1415-1422,1431-1438, 1447-1454, 1463-1470, 1479-1502, 1511-1518, 1527-1534,1543-1550, 1559-1566, 1575-1598, 1607-1614, 1623-1630, 1649-1656,1665-1682, 1691-1714, 1723-1730, 1739-1746, 1755-1762, 1771-1778,1787-1810, 1812, 1814, 1816, 1819, 1821, 1823, 1825, or 1828. In anotherembodiment, the sense region of interleukin and/or interleukin receptorconstructs comprises sequence having any of SEQ ID NOs. 1-81, 163-213,265-464, 665-735, 807-1029, 1253-1260, 1311-1318, 1327-1334, 1343-1350,1359-1366, 1375-1382, 1269-1276, 1407-1414, 1423-1430, 1439-1446,1455-1462, 1471-1478, 1277-1284, 1503-1510, 1519-1526, 1535-1542,1551-1558, 1567-1574, 1285-1292, 1599-1606, 1615-1622, 1631-1648,1657-1664, 1683-1690, 1303-1310, 1715-1722, 1731-1738, 1747-1754,1763-1770, 1779-1786, 1811, 1813, 1815, 1817, 1818, 1820, 1822, 1824,1826, or 1827.

In one embodiment, an siNA molecule of the invention comprises any ofSEQ ID NOs. 1-1828. The sequences shown in SEQ ID NOs: 1-1828 are notlimiting. An siNA molecule of the invention can comprise any contiguousinterleukin and/or interleukin receptor sequence (e.g., about 19 toabout 25, or about 19, 20, 21, 22, 23, 24, or 25 contiguous interleukinand/or interleukin receptor nucleotides).

In yet another embodiment, the invention features an siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in Table I.Chemical modifications in Tables III and IV and described herein can beapplied to any siNA construct of the invention.

In one embodiment of the invention an siNA molecule comprises anantisense strand having about 19 to about 29 (e.g., about 19, 20, 21,22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein the antisensestrand is complementary to a RNA sequence encoding a interleukin and/orinterleukin receptor protein, and wherein said siNA further comprises asense strand having about 19 to about 29 (e.g., about 19, 20, 21, 22,23, 24, 25, 26, 27, 28, or 29) nucleotides, and wherein said sensestrand and said antisense strand are distinct nucleotide sequences withat least about 19 complementary nucleotides.

In another embodiment of the invention an siNA molecule of the inventioncomprises an antisense region having about 19 to about 29 (e.g., about19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides, wherein theantisense region is complementary to a RNA sequence encoding ainterleukin and/or interleukin receptor protein, and wherein said siNAfurther comprises a sense region having about 19 to about 29 (e.g.,about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides,wherein said sense region and said antisense region comprise a linearmolecule with at least about 19 complementary nucleotides.

In one embodiment, an siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a interleukin and/orinterleukin receptor gene. Because interleukin and/or interleukinreceptor genes can share some degree of sequence homology with eachother, siNA molecules can be designed to target a class of interleukinand/or interleukin receptor genes or alternately specific interleukinand/or interleukin receptor genes (e.g., polymorphic variants) byselecting sequences that are either shared amongst different interleukinand/or interleukin receptor targets or alternatively that are unique fora specific interleukin and/or interleukin receptor target. Therefore, inone embodiment, the siNA molecule can be designed to target conservedregions of interleukin and/or interleukin receptor RNA sequences havinghomology among several interleukin and/or interleukin receptor genevariants so as to target a class of interleukin and/or interleukinreceptor genes with one siNA molecule. Accordingly, in one embodiment,the siNA molecule of the invention modulates the expression of one orboth interleukin and/or interleukin receptor alleles in a subject. Inanother embodiment, the siNA molecule can be designed to target asequence that is unique to a specific interleukin and/or interleukinreceptor RNA sequence (e.g., a single interleukin and/or interleukinreceptor allele or interleukin and/or interleukin receptor singlenucleotide polymorphism (SNP)) due to the high degree of specificitythat the siNA molecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplex nucleic acid moleculescontaining about 19 base pairs between oligonucleotides comprising about19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides.In yet another embodiment, siNA molecules of the invention compriseduplex nucleic acid molecules with overhanging ends of about 1 to about3 (e.g., about 1, 2, or 3) nucleotides, for example, about 21-nucleotideduplexes with about 19 base pairs and 3′-terminal mononucleotide,dinucleotide, or trinucleotide overhangs.

In one embodiment, the invention features one or morechemically-modified siNA constructs having specificity for interleukinand/or interleukin receptor expressing nucleic acid molecules, such asRNA encoding a interleukin and/or interleukin receptor protein.Non-limiting examples of such chemical modifications include withoutlimitation phosphorothioate internucleotide linkages,2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, “universal base” nucleotides, “acyclic” nucleotides,5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxyabasic residue incorporation. These chemical modifications, when used invarious siNA constructs, are shown to preserve RNAi activity in cellswhile at the same time, dramatically increasing the serum stability ofthese compounds. Furthermore, contrary to the data published by Parrishet al., supra, applicant demonstrates that multiple (greater than one)phosphorothioate substitutions are well-tolerated and confer substantialincreases in serum stability for modified siNA constructs.

In one embodiment, an siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, ansiNA molecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, an siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single-stranded, the percent modification can be based uponthe total number of nucleotides present in the single-stranded siNAmolecules. Likewise, if the siNA molecule is double-stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands.

One aspect of the invention features a double-stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of ainterleukin and/or interleukin receptor gene. In one embodiment, thedouble-stranded siNA molecule comprises one or more chemicalmodifications and each strand of the double-stranded siNA is about 21nucleotides long. In one embodiment, the double-stranded siNA moleculedoes not contain any ribonucleotides. In another embodiment, thedouble-stranded siNA molecule comprises one or more ribonucleotides. Inone embodiment, each strand of the double-stranded siNA moleculecomprises about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25,26, 27, 28, or 29) nucleotides, wherein each strand comprises about 19nucleotides that are complementary to the nucleotides of the otherstrand. In one embodiment, one of the strands of the double-strandedsiNA molecule comprises a nucleotide sequence that is complementary to anucleotide sequence or a portion thereof of the interleukin and/orinterleukin receptor gene, and the second strand of the double-strandedsiNA molecule comprises a nucleotide sequence substantially similar tothe nucleotide sequence of the interleukin and/or interleukin receptorgene or a portion thereof.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a interleukin and/or interleukin receptor gene comprising anantisense region, wherein the antisense region comprises a nucleotidesequence that is complementary to a nucleotide sequence of theinterleukin and/or interleukin receptor gene or a portion thereof, and asense region, wherein the sense region comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the interleukinand/or interleukin receptor gene or a portion thereof. In oneembodiment, the antisense region and the sense region each compriseabout 19 to about 23 (e.g. about 19, 20, 21, 22, or 23) nucleotides,wherein the antisense region comprises about 19 nucleotides that arecomplementary to nucleotides of the sense region.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a interleukin and/or interleukin receptor gene comprising a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by the interleukin and/or interleukin receptor gene or aportion thereof and the sense region comprises a nucleotide sequencethat is complementary to the antisense region.

In one embodiment, an siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, an siNA molecule comprising modifications described herein(e.g., comprising nucleotides having Formulae I-VII or siNA constructscomprising “Stab 00”-“Stab 25” (Table IV) or any combination thereof(see Table IV)) and/or any length described herein can comprise bluntends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended siNA molecule has anumber of base pairs equal to the number of nucleotides present in eachstrand of the siNA molecule. In another embodiment, the siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, the siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, an siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 18 to about 30nucleotides (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleotides). Other nucleotides present in a blunt ended siNAmolecule can comprise mismatches, bulges, loops, or wobble base pairs,for example, to modulate the activity of the siNA molecule to mediateRNA interference.

By “blunt ends” is meant symmetric termini or termini of adouble-stranded siNA molecule having no overhanging nucleotides. The twostrands of a double-stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a interleukin and/or interleukin receptor gene, wherein the siNAmolecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule. The sense regioncan be connected to the antisense region via a linker molecule, such asa polynucleotide linker or a non-nucleotide linker.

In one embodiment, the invention features double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a interleukin and/or interleukin receptor gene, wherein the siNAmolecule comprises about 19 to about 21 base pairs, and wherein eachstrand of the siNA molecule comprises one or more chemicalmodifications. In another embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of a interleukin and/orinterleukin receptor gene or a portion thereof, and the second strand ofthe double-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence or a portion thereof ofthe interleukin and/or interleukin receptor gene. In another embodiment,one of the strands of the double-stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of ainterleukin and/or interleukin receptor gene or portion thereof, and thesecond strand of the double-stranded siNA molecule comprises anucleotide sequence substantially similar to the nucleotide sequence orportion thereof of the interleukin and/or interleukin receptor gene. Inanother embodiment, each strand of the siNA molecule comprises about 19to about 23 nucleotides, and each strand comprises at least about 19nucleotides that are complementary to the nucleotides of the otherstrand. The interleukin and/or interleukin receptor gene can comprise,for example, sequences referred to in Table I.

In one embodiment, an siNA molecule of the invention comprises noribonucleotides. In another embodiment, an siNA molecule of theinvention comprises ribonucleotides.

In one embodiment, an siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a interleukin and/or interleukin receptorgene or a portion thereof, and the siNA further comprises a sense regioncomprising a nucleotide sequence substantially similar to the nucleotidesequence of the interleukin and/or interleukin receptor gene or aportion thereof. In another embodiment, the antisense region and thesense region each comprise about 19 to about 23 nucleotides and theantisense region comprises at least about 19 nucleotides that arecomplementary to nucleotides of the sense region. The interleukin and/orinterleukin receptor gene can comprise, for example, sequences referredto in Table I.

In one embodiment, an siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a interleukin and/or interleukin receptor gene, or aportion thereof, and the sense region comprises a nucleotide sequencethat is complementary to the antisense region. In one embodiment, thesiNA molecule is assembled from two separate oligonucleotide fragments,wherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule. In anotherembodiment, the sense region is connected to the antisense region via alinker molecule. In another embodiment, the sense region is connected tothe antisense region via a linker molecule, such as a nucleotide ornon-nucleotide linker. The interleukin and/or interleukin receptor genecan comprise, for example, sequences referred in to Table I.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a interleukin and/or interleukin receptor gene comprising a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by the interleukin and/or interleukin receptor gene or aportion thereof and the sense region comprises a nucleotide sequencethat is complementary to the antisense region, and wherein the siNAmolecule has one or more modified pyrimidine and/or purine nucleotides.In one embodiment, the pyrimidine nucleotides in the sense region are2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In another embodiment, the pyrimidinenucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In one embodiment, the pyrimidinenucleotides in the antisense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the antisense regionare 2′-O-methyl or 2′-deoxy purine nucleotides. In another embodiment ofany of the above-described siNA molecules, any nucleotides present in anon-complementary region of the sense strand (e.g. overhang region) are2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a interleukin and/or interleukin receptor gene, wherein the siNAmolecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule, and wherein thefragment comprising the sense region includes a terminal cap moiety atthe 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the fragment.In one embodiment, the terminal cap moiety is an inverted deoxy abasicmoiety or glyceryl moiety. In one embodiment, each of the two fragmentsof the siNA molecule comprise about 21 nucleotides.

In one embodiment, the invention features an siNA molecule comprising atleast one modified nucleotide, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide. The siNA can be, for example, of lengthbetween about 12 and about 36 nucleotides. In one embodiment, allpyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides. In one embodiment, the modified nucleotides inthe siNA include at least one 2′-deoxy-2′-fluoro cytidine or2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, themodified nucleotides in the siNA include at least one 2′-deoxy-2′-fluorocytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In oneembodiment, all uridine nucleotides present in the siNA are2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidinenucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In one embodiment, all adenosine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment,all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroguanosine nucleotides. The siNA can further comprise at least onemodified internucleotidic linkage, such as phosphorothioate linkage. Inone embodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a method of increasing thestability of an siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment,the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-deoxy-2′-fluoro cytidine and at least one2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridinenucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all cytidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In one embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a interleukin and/or interleukin receptor gene comprising a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by the interleukin and/or interleukin receptor gene or aportion thereof and the sense region comprises a nucleotide sequencethat is complementary to the antisense region, and wherein the purinenucleotides present in the antisense region comprise 2′-deoxy-purinenucleotides. In an alternative embodiment, the purine nucleotidespresent in the antisense region comprise 2′-O-methyl purine nucleotides.In either of the above embodiments, the antisense region can comprise aphosphorothioate internucleotide linkage at the 3′ end of the antisenseregion. Alternatively, in either of the above embodiments, the antisenseregion can comprise a glyceryl modification at the 3′ end of theantisense region. In another embodiment of any of the above-describedsiNA molecules, any nucleotides present in a non-complementary region ofthe antisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the antisense region of an siNA molecule of theinvention comprises sequence complementary to a portion of a interleukinand/or interleukin receptor transcript having sequence unique to aparticular interleukin and/or interleukin receptor disease relatedallele, such as sequence comprising a single nucleotide polymorphism(SNP) associated with the disease specific allele. As such, theantisense region of an siNA molecule of the invention can comprisesequence complementary to sequences that are unique to a particularallele to provide specificity in mediating selective RNAi against thedisease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a interleukin and/or interleukin receptor gene, wherein the siNAmolecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule. In anotherembodiment about 19 nucleotides of each fragment of the siNA moleculeare base-paired to the complementary nucleotides of the other fragmentof the siNA molecule and wherein at least two 3′ terminal nucleotides ofeach fragment of the siNA molecule are not base-paired to thenucleotides of the other fragment of the siNA molecule. In oneembodiment, each of the two 3′ terminal nucleotides of each fragment ofthe siNA molecule is a 2′-deoxy-pyrimidine nucleotide, such as a2′-deoxy-thymidine. In another embodiment, all 21 nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, about 19 nucleotides of the antisense region are base-pairedto the nucleotide sequence or a portion thereof of the RNA encoded bythe interleukin and/or interleukin receptor gene. In another embodiment,about 21 nucleotides of the antisense region are base-paired to thenucleotide sequence or a portion thereof of the RNA encoded by theinterleukin and/or interleukin receptor gene. In any of the aboveembodiments, the 5′-end of the fragment comprising said antisense regioncan optionally include a phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa interleukin and/or interleukin receptor RNA sequence (e.g., whereinsaid target RNA sequence is encoded by a interleukin and/or interleukinreceptor gene involved in the interleukin and/or interleukin receptorpathway), wherein the siNA molecule does not contain any ribonucleotidesand wherein each strand of the double-stranded siNA molecule is about 21nucleotides long. Examples of non-ribonucleotide containing siNAconstructs are combinations of stabilization chemistries shown in TableIV in any combination of Sense/Antisense chemistries, such as Stab 7/8,Stab 7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, or Stab18/20.

In one embodiment, the invention features a chemically synthesizeddouble-stranded RNA molecule that directs cleavage of a interleukinand/or interleukin receptor RNA via RNA interference, wherein eachstrand of said RNA molecule is about 21 to about 23 nucleotides inlength; one strand of the RNA molecule comprises nucleotide sequencehaving sufficient complementarity to the interleukin and/or interleukinreceptor RNA for the RNA molecule to direct cleavage of the interleukinand/or interleukin receptor RNA via RNA interference; and wherein atleast one strand of the RNA molecule comprises one or more chemicallymodified nucleotides described herein, such as deoxynucleotides,2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides,2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a medicament comprising ansiNA molecule of the invention.

In one embodiment, the invention features an active ingredientcomprising an siNA molecule of the invention.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to down-regulateexpression of a interleukin and/or interleukin receptor gene, whereinthe siNA molecule comprises one or more chemical modifications and eachstrand of the double-stranded siNA is about 18 to about 28 or more(e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 or more)nucleotides long.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits expressionof a interleukin and/or interleukin receptor gene, wherein one of thestrands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of interleukin and/or interleukin receptor RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of ainterleukin and/or interleukin receptor gene, wherein one of the strandsof the double-stranded siNA molecule is an antisense strand whichcomprises nucleotide sequence that is complementary to nucleotidesequence of interleukin and/or interleukin receptor RNA or a portionthereof, wherein the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand and wherein a majority of the pyrimidinenucleotides present in the double-stranded siNA molecule comprises asugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of ainterleukin and/or interleukin receptor gene, wherein one of the strandsof the double-stranded siNA molecule is an antisense strand whichcomprises nucleotide sequence that is complementary to nucleotidesequence of interleukin and/or interleukin receptor RNA that encodes aprotein or portion thereof, the other strand is a sense strand whichcomprises nucleotide sequence that is complementary to a nucleotidesequence of the antisense strand and wherein a majority of thepyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification. In one embodiment, each strand of thesiNA molecule comprises about 18 to about 29 or more (e.g., about 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 or more) nucleotides,wherein each strand comprises at least about 18 nucleotides that arecomplementary to the nucleotides of the other strand. In one embodiment,the siNA molecule is assembled from two oligonucleotide fragments,wherein one fragment comprises the nucleotide sequence of the antisensestrand of the siNA molecule and a second fragment comprises nucleotidesequence of the sense region of the siNA molecule. In one embodiment,the sense strand is connected to the antisense strand via a linkermolecule, such as a polynucleotide linker or a non-nucleotide linker. Ina further embodiment, the pyrimidine nucleotides present in the sensestrand are 2′-deoxy-2′fluoro pyrimidine nucleotides and the purinenucleotides present in the sense region are 2′-deoxy purine nucleotides.In another embodiment, the pyrimidine nucleotides present in the sensestrand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and the purinenucleotides present in the sense region are 2′-O-methyl purinenucleotides. In still another embodiment, the pyrimidine nucleotidespresent in the antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and any purine nucleotides present in the antisense strandare 2′-deoxy purine nucleotides. In another embodiment, the antisensestrand comprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotidesand one or more 2′-O-methyl purine nucleotides. In another embodiment,the pyrimidine nucleotides present in the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotidespresent in the antisense strand are 2′-O-methyl purine nucleotides. In afurther embodiment the sense strand comprises a 3′-end and a 5′-end,wherein a terminal cap moiety (e.g., an inverted deoxy abasic moiety orinverted deoxy nucleotide moiety such as inverted thymidine) is presentat the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sensestrand. In another embodiment, the antisense strand comprises aphosphorothioate internucleotide linkage at the 3′ end of the antisensestrand. In another embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of ainterleukin and/or interleukin receptor gene, wherein a majority of thepyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification, each of the two strands of the siNAmolecule can comprise about 21 nucleotides. In one embodiment, about 21nucleotides of each strand of the siNA molecule are base-paired to thecomplementary nucleotides of the other strand of the siNA molecule. Inanother embodiment, about 19 nucleotides of each strand of the siNAmolecule are base-paired to the complementary nucleotides of the otherstrand of the siNA molecule, wherein at least two 3′ terminalnucleotides of each strand of the siNA molecule are not base-paired tothe nucleotides of the other strand of the siNA molecule. In anotherembodiment, each of the two 3′ terminal nucleotides of each fragment ofthe siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine.In one embodiment, each strand of the siNA molecule is base-paired tothe complementary nucleotides of the other strand of the siNA molecule.In one embodiment, about 19 nucleotides of the antisense strand arebase-paired to the nucleotide sequence of the interleukin and/orinterleukin receptor RNA or a portion thereof. In one embodiment, about21 nucleotides of the antisense strand are base-paired to the nucleotidesequence of the interleukin and/or interleukin receptor RNA or a portionthereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of ainterleukin and/or interleukin receptor gene, wherein one of the strandsof the double-stranded siNA molecule is an antisense strand whichcomprises nucleotide sequence that is complementary to nucleotidesequence of interleukin and/or interleukin receptor RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the 5′-end of the antisense strand optionally includes aphosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of ainterleukin and/or interleukin receptor gene, wherein one of the strandsof the double-stranded siNA molecule is an antisense strand whichcomprises nucleotide sequence that is complementary to nucleotidesequence of interleukin and/or interleukin receptor RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the nucleotide sequence or a portion thereof of the antisensestrand is complementary to a nucleotide sequence of the untranslatedregion or a portion thereof of the interleukin and/or interleukinreceptor RNA.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of ainterleukin and/or interleukin receptor gene, wherein one of the strandsof the double-stranded siNA molecule is an antisense strand whichcomprises nucleotide sequence that is complementary to nucleotidesequence of interleukin and/or interleukin receptor RNA or a portionthereof, wherein the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand, wherein a majority of the pyrimidine nucleotidespresent in the double-stranded siNA molecule comprises a sugarmodification, and wherein the nucleotide sequence of the antisensestrand is complementary to a nucleotide sequence of the interleukinand/or interleukin receptor RNA or a portion thereof that is present inthe interleukin and/or interleukin receptor RNA.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention in a pharmaceutically acceptable carrieror diluent.

In a non-limiting example, the introduction of chemically-modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically-modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically-modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically-modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically-modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of an siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of an siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically-modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a RNA or DNA sequence encoding interleukinand/or interleukin receptor and the sense region can comprise sequencecomplementary to the antisense region. The siNA molecule can comprisetwo distinct strands having complementary sense and antisense regions.The siNA molecule can comprise a single strand having complementarysense and antisense regions.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against interleukin and/or interleukin receptorinside a cell or reconstituted in vitro system, wherein the chemicalmodification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) nucleotides comprising a backbone modifiedinternucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring orchemically-modified, each X and Y is independently O, S, N, alkyl, orsubstituted alkyl, each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl andwherein W, X, Y, and Z are optionally not all O. In another embodiment,a backbone modification of the invention comprises a phosphonoacetateand/or thiophosphonoacetate internucleotide linkage (see for exampleSheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically-modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically-modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically-modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, an siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically-modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against interleukin and/or interleukin receptorinside a cell or reconstituted in vitro system, wherein the chemicalmodification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) nucleotides or non-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to targetRNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotide or non-nucleotide of Formula II at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides ornon-nucleotides of Formula II at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotides or non-nucleotides of Formula II at the 3′-end of the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against interleukin and/or interleukin receptorinside a cell or reconstituted in vitro system, wherein the chemicalmodification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) nucleotides or non-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotide or non-nucleotide of Formula III at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) ornon-nucleotide(s) of Formula III at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotide or non-nucleotide of Formula III at the 3′-end of the sensestrand, the antisense strand, or both strands.

In another embodiment, an siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against interleukin and/or interleukin receptorinside a cell or reconstituted in vitro system, wherein the chemicalmodification comprises a 5′-terminal phosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, oracetyl; and wherein W, X, Y and Z are not all O.

In one embodiment, the invention features an siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features an siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of an siNA molecule of the invention,for example an siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against interleukin and/or interleukin receptorinside a cell or reconstituted in vitro system, wherein the chemicalmodification comprises one or more phosphorothioate internucleotidelinkages. For example, in a non-limiting example, the invention featuresa chemically-modified short interfering nucleic acid (siNA) having about1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkagesin one siNA strand. In yet another embodiment, the invention features achemically-modified short interfering nucleic acid (siNA) individuallyhaving about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioateinternucleotide linkages in both siNA strands. The phosphorothioateinternucleotide linkages can be present in one or both oligonucleotidestrands of the siNA duplex, for example in the sense strand, theantisense strand, or both strands. The siNA molecules of the inventioncan comprise one or more phosphorothioate internucleotide linkages atthe 3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sensestrand, the antisense strand, or both strands. For example, an exemplarysiNA molecule of the invention can comprise about 1 to about 5 or more(e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioateinternucleotide linkages at the 5′-end of the sense strand, theantisense strand, or both strands. In another non-limiting example, anexemplary siNA molecule of the invention can comprise one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands.

In one embodiment, the invention features an siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features an siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. Inanother embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends, being present in the same or differentstrand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5,specifically about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features an siNA moleculecomprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotidelinkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of one or both siNA sequence strands. In addition, the 2′-5′internucleotide linkage(s) can be present at various other positionswithin one or both siNA sequence strands, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of apyrimidine nucleotide in one or both strands of the siNA molecule cancomprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more including every internucleotide linkage of a purinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is about 18 to about 27(e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides inlength, wherein the duplex has about 18 to about 23 (e.g., about 18, 19,20, 21, 22, or 23) base pairs, and wherein the chemical modificationcomprises a structure having any of Formulae I-VII. For example, anexemplary chemically-modified siNA molecule of the invention comprises aduplex having two strands, one or both of which can bechemically-modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein each strand consists of about21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotideoverhang, and wherein the duplex has about 19 base pairs. In anotherembodiment, an siNA molecule of the invention comprises asingle-stranded hairpin structure, wherein the siNA is about 36 to about70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) basepairs, and wherein the siNA can include a chemical modificationcomprising a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically-modified siNA molecule ofthe invention comprises a linear oligonucleotide having about 42 toabout 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotidesthat is chemically-modified with a chemical modification having any ofFormulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 19 base pairs anda 2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.For example, a linear hairpin siNA molecule of the invention is designedsuch that degradation of the loop portion of the siNA molecule in vivocan generate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, an siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 23(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, or 23) base pairs and a 5′-terminal phosphate group thatcan be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In one embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 20 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20) base pairs, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically-modified siNA molecule of the invention comprises alinear oligonucleotide having about 25 to about 35 (e.g., about 25, 26,27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms an asymmetric hairpin structure having about 3 toabout 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, or 18) base pairs and a 5′-terminal phosphate group that can bechemically modified as described herein (for example a 5′-terminalphosphate group having Formula IV). In one embodiment, an asymmetrichairpin siNA molecule of the invention contains a stem loop motif,wherein the loop portion of the siNA molecule is biodegradable. Inanother embodiment, an asymmetric hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric double-stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 16 to about 25 (e.g., about 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides in length, wherein the sense region is about3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, or 18) nucleotides in length, wherein the sense region and theantisense region have at least 3 complementary nucleotides, and whereinthe siNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises an asymmetric double-stranded structure having separatepolynucleotide strands comprising sense and antisense regions, whereinthe antisense region is about 18 to about 22 (e.g., about 18, 19, 20,21, or 22) nucleotides in length and wherein the sense region is about 3to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double-stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, an siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) basepairs, and wherein the siNA can include a chemical modification, whichcomprises a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically-modified siNA molecule ofthe invention comprises a circular oligonucleotide having about 42 toabout 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotidesthat is chemically-modified with a chemical modification having any ofFormulae I-VII or any combination thereof, wherein the circularoligonucleotide forms a dumbbell shaped structure having about 19 basepairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; and R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R5, R3,R8 or R13 serves as a point of attachment to the siNA molecule of theinvention.

In another embodiment, an siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises Oand is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 10).

In another embodiment, a moiety having any of Formula V, VI or VII ofthe invention is at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of an siNA molecule of the invention. For example, a moietyhaving Formula V, VI or VII can be present at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the antisense strand, the sense strand, orboth antisense and sense strands of the siNA molecule. In addition, amoiety having Formula VII can be present at the 3′-end or the 5′-end ofa hairpin siNA molecule as described herein.

In another embodiment, an siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, an siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, an siNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides),wherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in thesense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) against interleukin and/or interleukinreceptor inside a cell or reconstituted in vitro system comprising asense region, wherein one or more pyrimidine nucleotides present in thesense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purinenucleotides present in the sense region are 2′-deoxy purine nucleotides(e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality of purine nucleotides are 2′-deoxy purinenucleotides), and an antisense region, wherein one or more pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and one or more purine nucleotides present in the antisense region are2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are2′-O-methyl purine nucleotides or alternately a plurality of purinenucleotides are 2′-O-methyl purine nucleotides). The sense region and/orthe antisense region can have a terminal cap modification, such as anymodification described herein or shown in FIG. 10, that is optionallypresent at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of thesense and/or antisense sequence. The sense and/or antisense region canoptionally further comprise a 3′-terminal nucleotide overhang havingabout 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. Theoverhang nucleotides can further comprise one or more (e.g., about 1, 2,3, 4 or more) phosphorothioate, phosphonoacetate, and/orthiophosphonoacetate internucleotide linkages. Non-limiting examples ofthese chemically-modified siNAs are shown in FIGS. 4 and 5 and TablesIII and IV herein. In any of these described embodiments, the purinenucleotides present in the sense region are alternatively 2′-O-methylpurine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methylpurine nucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides) and one or more purine nucleotidespresent in the antisense region are 2′-O-methyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotidesor alternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides). Also, in any of these embodiments, one or more purinenucleotides present in the sense region are alternatively purineribonucleotides (e.g., wherein all purine nucleotides are purineribonucleotides or alternately a plurality of purine nucleotides arepurine ribonucleotides) and any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Additionally, in any of these embodiments, one or more purinenucleotides present in the sense region and/or present in the antisenseregion are alternatively selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g.,wherein all purine nucleotides are selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides or alternately a plurality of purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a Northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE)nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides,2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methylnucleotides.

In one embodiment, the sense strand of a double-stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) against interleukin and/or interleukin receptorinside a cell or reconstituted in vitro system, wherein the chemicalmodification comprises a conjugate covalently attached to thechemically-modified siNA molecule. Non-limiting examples of conjugatescontemplated by the invention include conjugates and ligands describedin Vargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003,incorporated by reference herein in its entirety, including thedrawings. In another embodiment, the conjugate is covalently attached tothe chemically-modified siNA molecule via a biodegradable linker. In oneembodiment, the conjugate molecule is attached at the 3′-end of eitherthe sense strand, the antisense strand, or both strands of thechemically-modified siNA molecule. In another embodiment, the conjugatemolecule is attached at the 5′-end of either the sense strand, theantisense strand, or both strands of the chemically-modified siNAmolecule. In yet another embodiment, the conjugate molecule is attachedboth the 3′-end and 5′-end of either the sense strand, the antisensestrand, or both strands of the chemically-modified siNA molecule, or anycombination thereof. In one embodiment, a conjugate molecule of theinvention comprises a molecule that facilitates delivery of achemically-modified siNA molecule into a biological system, such as acell. In another embodiment, the conjugate molecule attached to thechemically-modified siNA molecule is a polyethylene glycol, human serumalbumin, or a ligand for a cellular receptor that can mediate cellularuptake. Examples of specific conjugate molecules contemplated by theinstant invention that can be attached to chemically-modified siNAmolecules are described in Vargeese et al., U.S. Ser. No. 10/201,394,filed Jul. 22, 2002 incorporated by reference herein. The type ofconjugates used and the extent of conjugation of siNA molecules of theinvention can be evaluated for improved pharmacokinetic profiles,bioavailability, and/or stability of siNA constructs while at the sametime maintaining the ability of the siNA to mediate RNAi activity. Assuch, one skilled in the art can screen siNA constructs that aremodified with various conjugates to determine whether the siNA conjugatecomplex possesses improved properties while maintaining the ability tomediate RNAi, for example in animal models as are generally known in theart.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotidelinker of the invention can be a linker of ≧2 nucleotides in length, forexample about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has a sequence that comprises a sequencerecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule where the target molecule does not naturally bind to a nucleicacid. The target molecule can be any molecule of interest. For example,the aptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemisty 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, an siNA molecule canbe assembled from a single oligonucleotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, an siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presence of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, an siNA molecule of the invention is asingle-stranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single-strandedpolynucleotide having complementarity to a target nucleic acid sequence.In another embodiment, the single-stranded siNA molecule of theinvention comprises a 5′-terminal phosphate group. In anotherembodiment, the single-stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a2′,3′-cyclic phosphate). In another embodiment, the single-stranded siNAmolecule of the invention comprises about 19 to about 29 (e.g., about19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. In yetanother embodiment, the single-stranded siNA molecule of the inventioncomprises one or more chemically modified nucleotides or non-nucleotidesdescribed herein. For example, all the positions within the siNAmolecule can include chemically-modified nucleotides such as nucleotideshaving any of Formulae I-VII, or any combination thereof to the extentthat the ability of the siNA molecule to support RNAi activity in a cellis maintained.

In one embodiment, an siNA molecule of the invention is asingle-stranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single-strandedpolynucleotide having complementarity to a target nucleic acid sequence,wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), anda terminal cap modification, such as any modification described hereinor shown in FIG. 10, that is optionally present at the 3′-end and/or the5′-end. The siNA optionally further comprises about 1 to about 4 or more(e.g., about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the3′-end of the siNA molecule, wherein the terminal nucleotides canfurther comprise one or more (e.g., 1, 2, 3, 4 or more)phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages, and wherein the siNA optionally furthercomprises a terminal phosphate group, such as a 5′-terminal phosphategroup. In any of these embodiments, any purine nucleotides present inthe antisense region are alternatively 2′-deoxy purine nucleotides(e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality of purine nucleotides are 2′-deoxy purinenucleotides). Also, in any of these embodiments, any purine nucleotidespresent in the siNA (i.e., purine nucleotides present in the senseand/or antisense region) can alternatively be locked nucleic acid (LNA)nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides oralternately a plurality of purine nucleotides are LNA nucleotides).Also, in any of these embodiments, any purine nucleotides present in thesiNA are alternatively 2′-methoxyethyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-methoxyethyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-methoxyethyl purinenucleotides). In another embodiment, any modified nucleotides present inthe single-stranded siNA molecules of the invention comprise modifiednucleotides having properties or characteristics similar to naturallyoccurring ribonucleotides. For example, the invention features siNAmolecules including modified nucleotides having a Northern conformation(e.g., Northern pseudorotation cycle, see for example Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Assuch, chemically modified nucleotides present in the single-strandedsiNA molecules of the invention are preferably resistant to nucleasedegradation while at the same time maintaining the capacity to mediateRNAi.

In one embodiment, the invention features a method for modulating theexpression of a interleukin and/or interleukin receptor gene within acell comprising: (a) synthesizing an siNA molecule of the invention,which can be chemically-modified, wherein one of the siNA strandscomprises a sequence complementary to RNA of the interleukin and/orinterleukin receptor gene; and (b) introducing the siNA molecule into acell under conditions suitable to modulate the expression of theinterleukin and/or interleukin receptor gene in the cell.

In one embodiment, the invention features a method for modulating theexpression of a interleukin and/or interleukin receptor gene within acell comprising: (a) synthesizing an siNA molecule of the invention,which can be chemically-modified, wherein one of the siNA strandscomprises a sequence complementary to RNA of the interleukin and/orinterleukin receptor gene and wherein the sense strand sequence of thesiNA comprises a sequence identical or substantially similar to thesequence of the target RNA; and (b) introducing the siNA molecule into acell under conditions suitable to modulate the expression of theinterleukin and/or interleukin receptor gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one interleukin and/or interleukin receptorgene within a cell comprising: (a) synthesizing siNA molecules of theinvention, which can be chemically-modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of the interleukinand/or interleukin receptor genes; and (b) introducing the siNAmolecules into a cell under conditions suitable to modulate theexpression of the interleukin and/or interleukin receptor genes in thecell.

In another embodiment, the invention features a method for modulatingthe expression of two or more interleukin and/or interleukin receptorgenes within a cell comprising: (a) synthesizing one or more siNAmolecules of the invention, which can be chemically-modified, whereinthe siNA strands comprise sequences complementary to RNA of theinterleukin and/or interleukin receptor genes and wherein the sensestrand sequences of the siNAs comprise sequences identical orsubstantially similar to the sequences of the target RNAs; and (b)introducing the siNA molecules into a cell under conditions suitable tomodulate the expression of the interleukin and/or interleukin receptorgenes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one interleukin and/or interleukin receptorgene within a cell comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of the interleukinand/or interleukin receptor gene and wherein the sense strand sequenceof the siNA comprises a sequence identical or substantially similar tothe sequences of the target RNAs; and (b) introducing the siNA moleculeinto a cell under conditions suitable to modulate the expression of theinterleukin and/or interleukin receptor genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients. In one embodiment, theinvention features a method of modulating the expression of ainterleukin and/or interleukin receptor gene in a tissue explantcomprising: (a) synthesizing an siNA molecule of the invention, whichcan be chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the interleukin and/or interleukinreceptor gene; and (b) introducing the siNA molecule into a cell of thetissue explant derived from a particular organism under conditionssuitable to modulate the expression of the interleukin and/orinterleukin receptor gene in the tissue explant. In another embodiment,the method further comprises introducing the tissue explant back intothe organism the tissue was derived from or into another organism underconditions suitable to modulate the expression of the interleukin and/orinterleukin receptor gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a interleukin and/or interleukin receptor gene in a tissueexplant comprising: (a) synthesizing an siNA molecule of the invention,which can be chemically-modified, wherein one of the siNA strandscomprises a sequence complementary to RNA of the interleukin and/orinterleukin receptor gene and wherein the sense strand sequence of thesiNA comprises a sequence identical or substantially similar to thesequence of the target RNA; and (b) introducing the siNA molecule into acell of the tissue explant derived from a particular organism underconditions suitable to modulate the expression of the interleukin and/orinterleukin receptor gene in the tissue explant. In another embodiment,the method further comprises introducing the tissue explant back intothe organism the tissue was derived from or into another organism underconditions suitable to modulate the expression of the interleukin and/orinterleukin receptor gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one interleukin and/or interleukin receptor genein a tissue explant comprising: (a) synthesizing siNA molecules of theinvention, which can be chemically-modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of the interleukinand/or interleukin receptor genes; and (b) introducing the siNAmolecules into a cell of the tissue explant derived from a particularorganism under conditions suitable to modulate the expression of theinterleukin and/or interleukin receptor genes in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the organism the tissue was derived from or intoanother organism under conditions suitable to modulate the expression ofthe interleukin and/or interleukin receptor genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a interleukin and/or interleukin receptor gene in anorganism comprising: (a) synthesizing an siNA molecule of the invention,which can be chemically-modified, wherein one of the siNA strandscomprises a sequence complementary to RNA of the interleukin and/orinterleukin receptor gene; and (b) introducing the siNA molecule intothe organism under conditions suitable to modulate the expression of theinterleukin and/or interleukin receptor gene in the organism. The levelof interleukin and/or interleukin receptor protein or RNA can bedetermined as is known in the art.

In another embodiment, the invention features a method of modulating theexpression of more than one interleukin and/or interleukin receptor genein an organism comprising: (a) synthesizing siNA molecules of theinvention, which can be chemically-modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of the interleukinand/or interleukin receptor genes; and (b) introducing the siNAmolecules into the organism under conditions suitable to modulate theexpression of the interleukin and/or interleukin receptor genes in theorganism. The level of interleukin and/or interleukin receptor proteinor RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating theexpression of a interleukin and/or interleukin receptor gene within acell comprising: (a) synthesizing an siNA molecule of the invention,which can be chemically-modified, wherein the siNA comprises asingle-stranded sequence having complementarity to RNA of theinterleukin and/or interleukin receptor gene; and (b) introducing thesiNA molecule into a cell under conditions suitable to modulate theexpression of the interleukin and/or interleukin receptor gene in thecell.

In another embodiment, the invention features a method for modulatingthe expression of more than one interleukin and/or interleukin receptorgene within a cell comprising: (a) synthesizing siNA molecules of theinvention, which can be chemically-modified, wherein the siNA comprisesa single-stranded sequence having complementarity to RNA of theinterleukin and/or interleukin receptor gene; and (b) contacting thecell in vitro or in vivo with the siNA molecule under conditionssuitable to modulate the expression of the interleukin and/orinterleukin receptor genes in the cell.

In one embodiment, the invention features a method of modulating theexpression of a interleukin and/or interleukin receptor gene in a tissueexplant comprising: (a) synthesizing an siNA molecule of the invention,which can be chemically-modified, wherein the siNA comprises asingle-stranded sequence having complementarity to RNA of theinterleukin and/or interleukin receptor gene; and (b) contacting thecell of the tissue explant derived from a particular organism with thesiNA molecule under conditions suitable to modulate the expression ofthe interleukin and/or interleukin receptor gene in the tissue explant.In another embodiment, the method further comprises introducing thetissue explant back into the organism the tissue was derived from orinto another organism under conditions suitable to modulate theexpression of the interleukin and/or interleukin receptor gene in thatorganism.

In another embodiment, the invention features a method of modulating theexpression of more than one interleukin and/or interleukin receptor genein a tissue explant comprising: (a) synthesizing siNA molecules of theinvention, which can be chemically-modified, wherein the siNA comprisesa single-stranded sequence having complementarity to RNA of theinterleukin and/or interleukin receptor gene; and (b) introducing thesiNA molecules into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate the expressionof the interleukin and/or interleukin receptor genes in the tissueexplant. In another embodiment, the method further comprises introducingthe tissue explant back into the organism the tissue was derived from orinto another organism under conditions suitable to modulate theexpression of the interleukin and/or interleukin receptor genes in thatorganism.

In one embodiment, the invention features a method of modulating theexpression of a interleukin and/or interleukin receptor gene in anorganism comprising: (a) synthesizing an siNA molecule of the invention,which can be chemically-modified, wherein the siNA comprises asingle-stranded sequence having complementarity to RNA of theinterleukin and/or interleukin receptor gene; and (b) introducing thesiNA molecule into the organism under conditions suitable to modulatethe expression of the interleukin and/or interleukin receptor gene inthe organism.

In another embodiment, the invention features a method of modulating theexpression of more than one interleukin and/or interleukin receptor genein an organism comprising: (a) synthesizing siNA molecules of theinvention, which can be chemically-modified, wherein the siNA comprisesa single-stranded sequence having complementarity to RNA of theinterleukin and/or interleukin receptor gene; and (b) introducing thesiNA molecules into the organism under conditions suitable to modulatethe expression of the interleukin and/or interleukin receptor genes inthe organism.

In one embodiment, the invention features a method of modulating theexpression of a interleukin and/or interleukin receptor gene in anorganism comprising contacting the organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of theinterleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing a disease, condition, trait, genotype or phenotype in asubject, comprising administering to the subject a composition of theinvention under conditions suitable for the treatment or prevention ofthe disease, condition, trait, genotype or phenotype in the subject,alone or in conjunction with one or more other therapeutic compounds. Inyet another embodiment, the invention features a method for reducing orpreventing tissue rejection in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thereduction or prevention of tissue rejection in the subject.

In one embodiment, the invention features a method for treating aninflammatory disease or condition in an organism comprising contactingthe organism with an siNA molecule of the invention under conditionssuitable to modulate the expression of the interleukin and/orinterleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing an allergic reaction, disease, or condition in an organismcomprising contacting the organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of theinterleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing an autoimmune disease or condition in an organism comprisingcontacting the organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the interleukin and/orinterleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing cancer in an organism comprising contacting the organism withan siNA molecule of the invention under conditions suitable to modulatethe expression of the interleukin and/or interleukin receptor gene inthe organism.

In one embodiment, the invention features a method for treating orpreventing a respiratory disease or condition in an organism comprisingcontacting the organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the interleukin and/orinterleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing a pulmonary disease or condition in an organism comprisingcontacting the organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the interleukin and/orinterleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing a neurodegenerative or neurological disease or condition inan organism comprising contacting the organism with an siNA molecule ofthe invention under conditions suitable to modulate the expression ofthe interleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing a proliferative disease or condition in an organismcomprising contacting the organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of theinterleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing a cardiovascular disease or condition in an organismcomprising contacting the organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of theinterleukin and/or interleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing a renal disease or condition in an organism comprisingcontacting the organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the interleukin and/orinterleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing a ocular disease or condition in an organism comprisingcontacting the organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the interleukin and/orinterleukin receptor gene in the organism.

In one embodiment, the invention features a method for treating orpreventing viral disease or infection in an organism comprisingcontacting the organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the interleukin and/orinterleukin receptor gene in the organism.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to treatdiseases or conditions discussed herein (e.g., cancers and otherproliferative conditions, viral infection, inflammatory disease,autoimmunity, respiratory disease, pulmonary disease, cardiovasculardisease, neurological disease, renal disease, ocular disease, etc.). Forexample, to treat a particular disease, condition, trait, genotype orphenotype, the siNA molecules can be administered to a subject or can beadministered to other appropriate cells evident to those skilled in theart, individually or in combination with one or more drugs underconditions suitable for the treatment.

In another embodiment, the invention features a method of modulating theexpression of more than one interleukin (e.g., any IL-1 through IL-27)and/or interleukin receptor (e.g., any IL-1R through IL-27R) genes in anorganism comprising contacting the organism with one or more siNAmolecules of the invention under conditions suitable to modulate theexpression of the interleukin and/or interleukin receptor genes in theorganism.

The siNA molecules of the invention can be designed to down regulate orinhibit target (e.g., interleukin and/or interleukin receptor) geneexpression through RNAi targeting of a variety of RNA molecules. In oneembodiment, the siNA molecules of the invention are used to targetvarious RNAs corresponding to a target gene. Non-limiting examples ofsuch RNAs include messenger RNA (mRNA), alternate RNA splice variants oftarget gene(s), post-transcriptionally modified RNA of target gene(s),pre-mRNA of target gene(s), and/or RNA templates. If alternate splicingproduces a family of transcripts that are distinguished by usage ofappropriate exons, the instant invention can be used to inhibit geneexpression through the appropriate exons to specifically inhibit or todistinguish among the functions of gene family members. For example, aprotein that contains an alternatively spliced transmembrane domain canbe expressed in both membrane bound and secreted forms. Use of theinvention to target the exon containing the transmembrane domain can beused to determine the functional consequences of pharmaceuticaltargeting of membrane bound as opposed to the secreted form of theprotein. Non-limiting examples of applications of the invention relatingto targeting these RNA molecules include therapeutic pharmaceuticalapplications, pharmaceutical discovery applications, moleculardiagnostic and gene function applications, and gene mapping, for exampleusing single nucleotide polymorphism mapping with siNA molecules of theinvention. Such applications can be implemented using known genesequences or from partial sequences available from an expressed sequencetag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as interleukin and/or interleukin receptor family genes.As such, siNA molecules targeting multiple interleukin and/orinterleukin receptor targets can provide increased therapeutic effect.In addition, siNA can be used to characterize pathways of gene functionin a variety of applications. For example, the present invention can beused to inhibit the activity of target gene(s) in a pathway to determinethe function of uncharacterized gene(s) in gene function analysis, mRNAfunction analysis, or translational analysis. The invention can be usedto determine potential target gene pathways involved in various diseasesand conditions toward pharmaceutical development. The invention can beused to understand pathways of gene expression involved in, for example,respiratory disease.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down regulate the expression of gene(s) that encode RNA referredto by Genbank Accession Nos., for example interleukin and/or interleukinreceptor genes encoding RNA sequence(s) referred to herein by GenbankAccession number, for example, Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 19 to about 25 (e.g., about 19, 20, 21,22, 23, 24, or 25) nucleotides in length. In one embodiment, the assaycan comprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. In another embodiment, fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, Northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4^(N), where N represents thenumber of base paired nucleotides in each of the siNA construct strands(e.g., for an siNA construct having 21 nucleotide sense and antisensestrands with 19 base pairs, the complexity would be 4¹⁹); and (b)assaying the siNA constructs of (a) above, under conditions suitable todetermine RNAi target sites within the target interleukin and/orinterleukin receptor RNA sequence. In another embodiment, the siNAmolecules of (a) have strands of a fixed length, for example about 23nucleotides in length. In yet another embodiment, the siNA molecules of(a) are of differing length, for example having strands of about 19 toabout 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described in Example 6 herein. In anotherembodiment, the assay can comprise a cell culture system in which targetRNA is expressed. In another embodiment, fragments of interleukin and/orinterleukin receptor RNA are analyzed for detectable levels of cleavage,for example by gel electrophoresis, Northern blot analysis, or RNAseprotection assays, to determine the most suitable target site(s) withinthe target interleukin and/or interleukin receptor RNA sequence. Thetarget interleukin and/or interleukin receptor RNA sequence can beobtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 19 to about25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. Inone embodiment, the assay can comprise a reconstituted in vitro siNAassay as described herein. In another embodiment, the assay can comprisea cell culture system in which target RNA is expressed. Fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, Northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by an siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention, which can be chemically-modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically-modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease or condition in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thediagnosis of the disease or condition in the subject. In anotherembodiment, the invention features a method for treating or preventing adisease or condition in a subject, comprising administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of the disease or condition in the subject,alone or in conjunction with one or more other therapeutic compounds. Inyet another embodiment, the invention features a method for reducing orpreventing, for example, respiratory disease (e.g., asthma) in asubject, comprising administering to the subject a composition of theinvention under conditions suitable for the reduction or prevention ofthe respiratory disease in the subject.

In another embodiment, the invention features a method for validating ainterleukin and/or interleukin receptor gene target, comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands includes a sequencecomplementary to RNA of a interleukin and/or interleukin receptor targetgene; (b) introducing the siNA molecule into a cell, tissue, or organismunder conditions suitable for modulating expression of the interleukinand/or interleukin receptor target gene in the cell, tissue, ororganism; and (c) determining the function of the gene by assaying forany phenotypic change in the cell, tissue, or organism.

In another embodiment, the invention features a method for validating ainterleukin and/or interleukin receptor target comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands includes a sequencecomplementary to RNA of a interleukin and/or interleukin receptor targetgene; (b) introducing the siNA molecule into a biological system underconditions suitable for modulating expression of the interleukin and/orinterleukin receptor target gene in the biological system; and (c)determining the function of the gene by assaying for any phenotypicchange in the biological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human oranimal, wherein the system comprises the components required for RNAiactivity. The term “biological system” includes, for example, a cell,tissue, or organism, or extract thereof. The term biological system alsoincludes reconstituted RNAi systems that can be used in an in vitrosetting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing an siNAmolecule of the invention, which can be chemically-modified, that can beused to modulate the expression of a interleukin and/or interleukinreceptor target gene in a biological system, including, for example, ina cell, tissue, or organism. In another embodiment, the inventionfeatures a kit containing more than one siNA molecule of the invention,which can be chemically-modified, that can be used to modulate theexpression of more than one interleukin and/or interleukin receptortarget gene in a biological system, including, for example, in a cell,tissue, or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically-modified. Inanother embodiment, the cell containing an siNA molecule of theinvention is a mammalian cell. In yet another embodiment, the cellcontaining an siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of an siNA molecule of the invention,which can be chemically-modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble-stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing ansiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand.

In one embodiment, cleavage of the linker molecule in (c) above takesplace during deprotection of the oligonucleotide, for example underhydrolysis conditions using an alkylamine base such as methylamine. Inone embodiment, the method of synthesis comprises solid phase synthesison a solid support such as controlled pore glass (CPG) or polystyrene,wherein the first sequence of (a) is synthesized on a cleavable linker,such as a succinyl linker, using the solid support as a scaffold. Thecleavable linker in (a) used as a scaffold for synthesizing the secondstrand can comprise similar reactivity as the solid support derivatizedlinker, such that cleavage of the solid support derivatized linker andthe cleavable linker of (a) takes place concomitantly. In anotherembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group, which can be employed in a trityl-on synthesisstrategy as described herein. In yet another embodiment, the chemicalmoiety, such as a dimethoxytrityl group, is removed during purification,for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizingan siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double-stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siNA molecule, for example using a trityl-on synthesisstrategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against interleukin and/or interleukin receptor, wherein the siNAconstruct comprises one or more chemical modifications, for example, oneor more chemical modifications having any of Formulae I-VII or anycombination thereof that increases the nuclease resistance of the siNAconstruct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In one embodiment, the invention features siNA constructs that mediateRNAi against interleukin and/or interleukin receptor, wherein the siNAconstruct comprises one or more chemical modifications described hereinthat modulates the binding affinity between the sense and antisensestrands of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formula I-VII or any combination thereof intoan siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against interleukin and/or interleukin receptor, wherein the siNAconstruct comprises one or more chemical modifications described hereinthat modulates the binding affinity between the antisense strand of thesiNA construct and a complementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against interleukin and/or interleukin receptor, wherein the siNAconstruct comprises one or more chemical modifications described hereinthat modulates the binding affinity between the antisense strand of thesiNA construct and a complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against interleukin and/or interleukin receptor, wherein the siNAconstruct comprises one or more chemical modifications described hereinthat modulate the polymerase activity of a cellular polymerase capableof generating additional endogenous siNA molecules having sequencehomology to the chemically-modified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically-modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically-modified siNAmolecule.

In one embodiment, the invention features chemically-modified siNAconstructs that mediate RNAi against interleukin and/or interleukinreceptor in a cell, wherein the chemical modifications do notsignificantly effect the interaction of siNA with a target RNA molecule,DNA molecule and/or proteins or other factors that are essential forRNAi in a manner that would decrease the efficacy of RNAi mediated bysuch siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against interleukin and/orinterleukin receptor comprising (a) introducing nucleotides having anyof Formula I-VII or any combination thereof into an siNA molecule, and(b) assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity againstinterleukin and/or interleukin receptor target RNA comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingimproved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity againstinterleukin and/or interleukin receptor target DNA comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingimproved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against interleukin and/or interleukin receptor, wherein the siNAconstruct comprises one or more chemical modifications described hereinthat modulates the cellular uptake of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules against interleukin and/or interleukin receptor withimproved cellular uptake comprising (a) introducing nucleotides havingany of Formula I-VII or any combination thereof into an siNA molecule,and (b) assaying the siNA molecule of step (a) under conditions suitablefor isolating siNA molecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against interleukin and/or interleukin receptor, wherein the siNAconstruct comprises one or more chemical modifications described hereinthat increases the bioavailability of the siNA construct, for example,by attaching polymeric conjugates such as polyethyleneglycol orequivalent conjugates that improve the pharmacokinetics of the siNAconstruct, or by attaching conjugates that target specific tissue typesor cell types in vivo. Non-limiting examples of such conjugates aredescribed in Vargeese et al., U.S. Ser. No. 10/201,394 incorporated byreference herein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability, comprising (a)introducing a conjugate into the structure of an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; polyamines, such as spermine or spermidine;and others.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or be recognized by cellular proteinsthat facilitate RNAi.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. Such design or modifications are expected to enhance theactivity of siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In one embodiment, the terminalcap moiety comprises an inverted abasic, inverted deoxy abasic, invertednucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkylgroup, a heterocycle, or any other group that prevents RNAi activity inwhich the second sequence serves as a guide sequence or template forRNAi.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In one embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.10, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of an siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 10(e.g. inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g. the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, an siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding targetRNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”,“Stab 23/24”, and “Stab 24/25” chemistries and variants thereof (seeTable IV) wherein the 5′-end and 3′-end of the sense strand of the siNAdo not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising introducing oneor more chemical modifications into the structure of an siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In one embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”, and“Stab 24/25” chemistries and variants thereof (see Table IV) wherein the5′-end and 3′-end of the sense strand of the siNA do not comprise ahydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNAmolecules that are active in mediating RNA interference against a targetnucleic acid sequence comprising (a) generating a plurality ofunmodified siNA molecules, (b) screening the siNA molecules of step (a)under conditions suitable for isolating siNA molecules that are activein mediating RNA interference against the target nucleic acid sequence,and (c) introducing chemical modifications (e.g. chemical modificationsas described herein or as otherwise known in the art) into the activesiNA molecules of (b). In one embodiment, the method further comprisesre-screening the chemically modified siNA molecules of step (c) underconditions suitable for isolating chemically modified siNA moleculesthat are active in mediating RNA interference against the target nucleicacid sequence.

In one embodiment, the invention features a method for screeningchemically modified siNA molecules that are active in mediating RNAinterference against a target nucleic acid sequence comprising (a)generating a plurality of chemically modified siNA molecules (e.g. siNAmolecules as described herein or as otherwise known in the art), and (b)screening the siNA molecules of step (a) under conditions suitable forisolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter, that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include an siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in FIGS. 4-6, and Tables IIand III herein. For example the siNA can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e. each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex ordouble-stranded structure, for example wherein the double-strandedregion is about 19 base pairs); the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. Alternatively, the siNA is assembled froma single oligonucleotide, where the self-complementary sense andantisense regions of the siNA are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s). The siNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siNA molecule capable of mediating RNAi. The siNA canalso comprise a single-stranded polynucleotide having nucleotidesequence complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof (for example, where such siNA moleculedoes not require the presence within the siNA molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single-stranded polynucleotide can furthercomprise a terminal phosphate group, such as a 5′-phosphate (see forexample Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al.,2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siNA molecules ofthe invention can be used to epigenetically silence genes at both thepost-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation pattern to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237).

In one embodiment, an siNA molecule of the invention is a duplex formingoligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al.,U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and McSwiggen et al.,PCT/US04/16390, filed May 24, 2004).

In one embodiment, an siNA molecule of the invention is amultifunctional siNA, (see for example FIGS. 16-22 and Jadhav et al.,U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and McSwiggen et al.,PCT/US04/16390, filed May 24, 2004). The multifunctional siNA of theinvention can comprise sequence targeting, for example, two regions ofinterleukin and/or interleukin receptor RNA (see for example targetsequences in Tables II and III).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 19 to about 22, or about 19, 20, 21, or 22 nucleotides) anda loop region comprising about 4 to about 8 (e.g., about 4, 5, 6, 7, or8) nucleotides, and a sense region having about 3 to about 18 (e.g.,about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18)nucleotides that are complementary to the antisense region. Theasymmetric hairpin siNA molecule can also comprise a 5′-terminalphosphate group that can be chemically modified. The loop portion of theasymmetric hairpin siNA molecule can comprise nucleotides,non-nucleotides, linker molecules, or conjugate molecules as describedherein.

By “asymmetric duplex” as used herein is meant an siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system e.g. about 19 to about 22 (e.g. about 19, 20,21, or 22) nucleotides and a sense region having about 3 to about 18(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18)nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence.

By “gene”, or “target gene”, is meant, a nucleic acid that encodes anRNA, for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules for siNAmediated RNA interference in modulating the activity of fRNA or ncRNAinvolved in functional or regulatory cellular processes. Aberrant fRNAor ncRNA activity leading to disease can therefore be modulated by siNAmolecules of the invention. siNA molecules targeting fRNA and ncRNA canalso be used to manipulate or alter the genotype or phenotype of anorganism or cell, by intervening in cellular processes such as geneticimprinting, transcription, translation, or nucleic acid processing(e.g., transamination, methylation etc.). The target gene can be a genederived from a cell, an endogenous gene, a transgene, or exogenous genessuch as genes of a pathogen, for example a virus, which is present inthe cell after infection thereof. The cell containing the target genecan be derived from or contained in any organism, for example a plant,animal, protozoan, virus, bacterium, or fungus. Non-limiting examples ofplants include monocots, dicots, or gymnosperms. Non-limiting examplesof animals include vertebrates or invertebrates. Non-limiting examplesof fungi include molds or yeasts. For a review, see for example Snyderand Gerstein, 2003, Science, 300, 258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair,such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N-3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl,GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-aminosymmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3,AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AUamino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CCcarbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GAN3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GGamino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GUcarbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl,UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H—N3,GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “interleukin” is meant, any interleukin (e.g., IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24,IL-25, IL-26, and IL-27) polypeptide, protein and/or a polynucleotideencoding an interleukin protein, peptide, or portion thereof (such aspolynucleotides referred to by Genbank Accession numbers in Table I orany other interleukin transcript derived from an interleukin gene). Theterm “interleukin” is also meant to include other interleukin encodingsequence, such as mutant interleukin genes, splice variants ofinterleukin genes, and interleukin gene polymorphisms, such as thoseassociated with a disease, trait, or condition.

By “interleukin protein” is meant, any interleukin peptide or protein ora component thereof, wherein the peptide or protein is encoded by aninterleukin gene or having interleukin activity.

By “interleukin receptor” is meant, any interleukin receptor (e.g.,IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R,IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R,IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26R, and IL-27R)polypeptide, protein and/or a polynucleotide encoding an interleukinreceptor protein, peptide, or portion thereof (such as polynucleotidesreferred to by Genbank Accession numbers in Table I or any otherinterleukin receptor transcript derived from an interleukin receptorgene). The term “interleukin receptor” is also meant to include otherinterleukin receptor encoding sequence, such as mutant interleukinreceptor genes, splice variants of interleukin receptor genes, andinterleukin receptor gene polymorphisms, such as those associated with adisease, trait, or condition.

By “interleukin receptor protein” is meant, any interleukin receptorpeptide or protein or a component thereof, wherein the peptide orprotein is encoded by an interleukin receptor gene or having interleukinreceptor activity.

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system or organism to anotherbiological system or organism. The polynucleotide can include bothcoding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of an siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of an siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence.

In one embodiment, siNA molecules of the invention that down regulate orreduce interleukin and/or interleukin receptor gene expression are usedfor preventing or reducing cancers and other proliferative conditions,viral infection, inflammatory disease, autoimmunity, respiratorydisease, pulmonary disease, cardiovascular disease, neurologicaldisease, renal disease, ocular disease, liver disease, mitochondrialdisease, endocrine disease, prion disease, reproduction related diseasesand conditions or any other disease associated with interleukin and/orinterleukin receptor gene expression in a subject. In one embodiment,the siNA molecules of the invention that down regulate or reduceinterleukin and/or interleukin receptor gene expression are used fortreating or preventing asthma, chronic obstructive pulmonary disease or“COPD”, allergic rhinitis, sinusitis, pulmonary vasoconstriction,inflammation, allergies, impeded respiration, respiratory distresssyndrome, cystic fibrosis, pulmonary hypertension, pulmonaryvasoconstriction, or emphysema in a subject.

By “cancer” is meant a group of diseases characterized by uncontrolledgrowth and spread of abnormal cells.

By “proliferative disease” or “cancer” is meant, any disease, condition,trait, genotype or phenotype characterized by unregulated cell growth orreplication as is known in the art; including AIDS related cancers suchas Kaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and any other cancer or proliferative disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “inflammatory disease” or “inflammatory condition” is meant anydisease, condition, trait, genotype or phenotype characterized by aninflammatory or allergic process as is known in the art, such asinflammation, acute inflammation, chronic inflammation, atherosclerosis,restenosis, asthma, allergic rhinitis, atopic dermatitis, psoriasis,septic shock, rheumatoid arthritis, inflammatory bowl disease,inflammatory pelvic disease, pain, ocular inflammatory disease, celiacdisease, Leigh Syndrome, Glycerol Kinase Deficiency, Familialeosinophilia (FE), autosomal recessive spastic ataxia, laryngealinflammatory disease; Tuberculosis, Chronic cholecystitis,Bronchiectasis, Silicosis and other pneumoconiosis, and any otherinflammatory disease, condition, trait, genotype or phenotype that canrespond to the modulation of disease related gene expression in a cellor tissue, alone or in combination with other therapies.

By “respiratory disease” is meant, any disease or condition affectingthe respiratory tract, such as asthma, chronic obstructive pulmonarydisease or “COPD”, allergic rhinitis, sinusitis, pulmonaryvasoconstriction, inflammation, allergies, impeded respiration,respiratory distress syndrome, cystic fibrosis, pulmonary hypertension,pulmonary vasoconstriction, emphysema, and any other respiratorydisease, condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “autoimmune disease” or “autoimmune condition” is meant, any disease,condition, trait, genotype or phenotype characterized by autoimmunity asis known in the art, such as multiple sclerosis, diabetes mellitus,lupus, celiac disease, Crohn's disease, ulcerative colitis,Guillain-Barre syndrome, scleroderms, Goodpasture's syndrome, Wegener'sgranulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primarybiliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis,Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier'ssyndrome; transplantation rejection (e.g., prevention of allograftrejection) pernicious anemia, rheumatoid arthritis, systemic lupuserythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus,multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave'sdisease, and any other autoimmune disease, condition, trait, genotype orphenotype that can respond to the modulation of disease related geneexpression in a cell or tissue, alone or in combination with othertherapies.

By “neurological disease” or “neurological disease” is meant anydisease, disorder, or condition affecting the central or peripheralnervous system, including ADHD, AIDS—Neurological Complications, Absenceof the Septum Pellucidum, Acquired Epileptiform Aphasia, AcuteDisseminated Encephalomyelitis, Adrenoleukodystrophy, Agenesis of theCorpus Callosum, Agnosia, Aicardi Syndrome, Alexander Disease, Alpers'Disease, Alternating Hemiplegia, Alzheimer's Disease, AmyotrophicLateral Sclerosis, Anencephaly, Aneurysm, Angelman Syndrome,Angiomatosis, Anoxia, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis,Arnold-Chiari Malformation, Arteriovenous Malformation, Aspartame,Asperger Syndrome, Ataxia Telangiectasia, Ataxia, AttentionDeficit-Hyperactivity Disorder, Autism, Autonomic Dysfunction, BackPain, Barth Syndrome, Batten Disease, Behcet's Disease, Bell's Palsy,Benign Essential Blepharospasm, Benign Focal Amyotrophy, BenignIntracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger'sDisease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus BirthInjuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, BrainAneurysm, Brain Injury, Brain and Spinal Tumors, Brown-Sequard Syndrome,Bulbospinal Muscular Atrophy, Canavan Disease, Carpal Tunnel Syndrome,Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation,Central Cervical Cord Syndrome, Central Cord Syndrome, Central PainSyndrome, Cephalic Disorders, Cerebellar Degeneration, CerebellarHypoplasia, Cerebral Aneurysm, Cerebral Arteriosclerosis, CerebralAtrophy, Cerebral Beriberi, Cerebral Gigantism, Cerebral Hypoxia,Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome,Charcot-Marie-Tooth Disorder, Chiari Malformation, Chorea,Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy(CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne SyndromeType II, Coffin Lowry Syndrome, Coma, including Persistent VegetativeState, Complex Regional Pain Syndrome, Congenital Facial Diplegia,Congenital Myasthenia, Congenital Myopathy, Congenital VascularCavernous Malformations, Corticobasal Degeneration, Cranial Arteritis,Craniosynostosis, Creutzfeldt-Jakob Disease, Cumulative TraumaDisorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease(CIBD), Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome,Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome,Dejerine-Klumpke Palsy, Dementia—Multi-Infarct, Dementia—Subcortical,Dementia With Lewy Bodies, Dermatomyositis, Developmental Dyspraxia,Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet'sSyndrome, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia,Dystonias, Early Infantile Epileptic Encephalopathy, Empty SellaSyndrome, Encephalitis Lethargica, Encephalitis and Meningitis,Encephaloceles, Encephalopathy, Encephalotrigeminal Angiomatosis,Epilepsy, Erb's Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies,Fabry's Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia,Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification,Familial Spastic Paralysis, Febrile Seizures (e.g., GEFS and GEFS plus),Fisher Syndrome, Floppy Infant Syndrome, Friedreich's Ataxia, Gaucher'sDisease, Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease,Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid CellLeukodystrophy, Glossopharyngeal Neuralgia, Guillain-Barre Syndrome,HTLV-1 Associated Myelopathy, Hallervorden-Spatz Disease, Head Injury,Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans,Hereditary Neuropathies, Hereditary Spastic Paraplegia, HeredopathiaAtactica Polyneuritiformis, Herpes Zoster Oticus, Herpes Zoster,Hirayama Syndrome, Holoprosencephaly, Huntington's Disease,Hydranencephaly, Hydrocephalus—Normal Pressure, Hydrocephalus,Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia,Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis,Incontinentia Pigmenti, Infantile Hypotonia, Infantile Phytanic AcidStorage Disease, Infantile Refsum Disease, Infantile Spasms,Inflammatory Myopathy, Intestinal Lipodystrophy, Intracranial Cysts,Intracranial Hypertension, Isaac's Syndrome, Joubert Syndrome,Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome,Kleine-Levin syndrome, Klippel Feil Syndrome, Klippel-Trenaunay Syndrome(KTS), Kluver-Bucy Syndrome, Korsakoff's Amnesic Syndrome, KrabbeDisease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton MyasthenicSyndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous NerveEntrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh'sDisease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy,Levine-Critchley Syndrome, Lewy Body Dementia, Lissencephaly, Locked-InSyndrome, Lou Gehrig's Disease, Lupus—Neurological Sequelae, LymeDisease—Neurological Complications, Machado-Joseph Disease,Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome,Meningitis, Menkes Disease, Meralgia Paresthetica, MetachromaticLeukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome,Mini-Strokes, Mitochondrial Myopathies, Mobius Syndrome, MonomelicAmyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses,Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal MotorNeuropathy, Multiple Sclerosis, Multiple System Atrophy with OrthostaticHypotension, Multiple System Atrophy, Muscular Dystrophy,Myasthenia—Congenital, Myasthenia Gravis, Myelinoclastic DiffuseSclerosis, Myoclonic Encephalopathy of Infants, Myoclonus,Myopathy—Congenital, Myopathy—Thyrotoxic, Myopathy, Myotonia Congenita,Myotonia, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with BrainIron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome,Neurological Complications of AIDS, Neurological Manifestations of PompeDisease, Neuromyelitis Optica, Neuromyotonia, Neuronal CeroidLipofuscinosis, Neuronal Migration Disorders, Neuropathy—Hereditary,Neurosarcoidosis, Neurotoxicity, Nevus Cavemosus, Niemann-Pick Disease,O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Occult SpinalDysraphism Sequence, Ohtahara Syndrome, Olivopontocerebellar Atrophy,Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome,Pain—Chronic, Paraneoplastic Syndromes, Paresthesia, Parkinson'sDisease, Parmyotonia Congenita, Paroxysmal Choreoathetosis, ParoxysmalHemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir IISyndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy,Periventricular Leukomalacia, Persistent Vegetative State, PervasiveDevelopmental Disorders, Phytanic Acid Storage Disease, Pick's Disease,Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease,Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia,Postinfectious Encephalomyelitis, Postural Hypotension, PosturalOrthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, PrimaryLateral Sclerosis, Prion Diseases, Progressive Hemifacial Atrophy,Progressive Locomotor Ataxia, Progressive MultifocalLeukoencephalopathy, Progressive Sclerosing Poliodystrophy, ProgressiveSupranuclear Palsy, Pseudotumor Cerebri, Pyridoxine Dependent andPyridoxine Responsive Siezure Disorders, Ramsay Hunt Syndrome Type I,Ramsay Hunt Syndrome Type II, Rasmussen's Encephalitis and otherautoimmune epilepsies, Reflex Sympathetic Dystrophy Syndrome, RefsumDisease—Infantile, Refsum Disease, Repetitive Motion Disorders,Repetitive Stress Injuries, Restless Legs Syndrome,Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome,Riley-Day Syndrome, SUNCT Headache, Sacral Nerve Root Cysts, Saint VitusDance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease,Schizencephaly, Seizure Disorders, Septo-Optic Dysplasia, SevereMyoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles,Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, Sleeping Sickness,Soto's Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction,Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy,Spinocerebellar Atrophy, Steele-Richardson-Olszewski Syndrome,Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-WeberSyndrome, Subacute Sclerosing Panencephalitis, SubcorticalArteriosclerotic Encephalopathy, Swallowing Disorders, Sydenham Chorea,Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia,Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, TarlovCysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal CordSyndrome, Thomsen Disease, Thoracic Outlet Syndrome, ThyrotoxicMyopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, TransientIschemic Attack, Transmissible Spongiform Encephalopathies, TransverseMyelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, TropicalSpastic Paraparesis, Tuberous Sclerosis, Vascular Erectile Tumor,Vasculitis including Temporal Arteritis, Von Economo's Disease, VonHippel-Lindau disease (VHL), Von Recklinghausen's Disease, Wallenberg'sSyndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, WestSyndrome, Whipple's Disease, Williams Syndrome, Wilson's Disease,X-Linked Spinal and Bulbar Muscular Atrophy, and Zellweger Syndrome.

By “infectious disease” is meant any disease, condition, trait, genotypeor phenotype associated with an infectious agent, such as a virus,bacteria, fungus, prion, or parasite. Non-limiting examples of variousviral genes that can be targeted using siNA molecules of the inventioninclude Hepatitis C Virus (HCV, for example Genbank Accession Nos:D11168, D50483.1, L38318 and S82227), Hepatitis B Virus (HBV, forexample GenBank Accession No. AF100308.1), Human Immunodeficiency Virustype 1 (HIV-1, for example GenBank Accession No. U51188), HumanImmunodeficiency Virus type 2 (HIV-2, for example GenBank Accession No.X60667), West Nile Virus (WNV for example GenBank accession No.NC_(—)001563), cytomegalovirus (CMV for example GenBank Accession No.NC_(—)001347), respiratory syncytial virus (RSV for example GenBankAccession No. NC_(—)001781), influenza virus (for example GenBankAccession No. AF037412, rhinovirus (for example, GenBank accessionnumbers: D00239, X02316, X01087, L24917, M16248, K02121, X01087),papillomavirus (for example GenBank Accession No. NC_(—)001353), HerpesSimplex Virus (HSV for example GenBank Accession No. NC_(—)001345), andother viruses such as HTLV (for example GenBank Accession No.AJ430-458). Due to the high sequence variability of many viral genomes,selection of siNA molecules for broad therapeutic applications wouldlikely involve the conserved regions of the viral genome. Nonlimitingexamples of conserved regions of the viral genomes include but are notlimited to 5′-Non Coding Regions (NCR), 3′-Non Coding Regions (NCR)and/or internal ribosome entry sites (IRES). siNA molecules designedagainst conserved regions of various viral genomes will enable efficientinhibition of viral replication in diverse patient populations and mayensure the effectiveness of the siNA molecules against viral quasispecies which evolve due to mutations in the non-conserved regions ofthe viral genome. Non-limiting examples of bacterial infections includeActinomycosis, Anthrax, Aspergillosis, Bacteremia, Bacterial Infectionsand Mycoses, Bartonella Infections, Botulism, Brucellosis, BurkholderiaInfections, Campylobacter Infections, Candidiasis, Cat-Scratch Disease,Chlamydia Infections, Cholera, Clostridium Infections,Coccidioidomycosis, Cross Infection, Cryptococcosis, Dermatomycoses,Dermatomycoses, Diphtheria, Ehrlichiosis, Escherichia coli Infections,Fasciitis, Necrotizing, Fusobacterium Infections, Gas Gangrene,Gram-Negative Bacterial Infections, Gram-Positive Bacterial Infections,Histoplasmosis, Impetigo, Klebsiella Infections, Legionellosis, Leprosy,Leptospirosis, Listeria Infections, Lyme Disease, Maduromycosis,Melioidosis, Mycobacterium Infections, Mycoplasma Infections, Mycoses,Nocardia Infections, Onychomycosis, Ornithosis, Plague, PneumococcalInfections, Pseudomonas Infections, Q Fever, Rat-Bite Fever, RelapsingFever, Rheumatic Fever, Rickettsia Infections, Rocky Mountain SpottedFever, Salmonella Infections, Scarlet Fever, Scrub Typhus, Sepsis,Sexually Transmitted Diseases—Bacterial, Bacterial Skin Diseases,Staphylococcal Infections, Streptococcal Infections, Tetanus, Tick-BorneDiseases, Tuberculosis, Tularemia, Typhoid Fever, Typhus, EpidemicLouse-Borne, Vibrio Infections, Yaws, Yersinia Infections, Zoonoses, andZygomycosis. Non-limiting examples of fungal infections includeAspergillosis, Blastomycosis, Coccidioidomycosis, Cryptococcosis, FungalInfections of Fingernails and Toenails, Fungal Sinusitis,Histoplasmosis, Histoplasmosis, Mucormycosis, Nail Fungal Infection,Paracoccidioidomycosis, Sporotrichosis, Valley Fever(Coccidioidomycosis), and Mold Allergy.

By “ocular disease” is meant, any disease, condition, trait, genotype orphenotype of the eye and related structures, such as Cystoid MacularEdema, Asteroid Hyalosis, Pathological Myopia and Posterior Staphyloma,Toxocariasis (Ocular Larva Migrans), Retinal Vein Occlusion, PosteriorVitreous Detachment, Tractional Retinal Tears, Epiretinal Membrane,Diabetic Retinopathy, Lattice Degeneration, Retinal Vein Occlusion,Retinal Artery Occlusion, Macular Degeneration (e.g., age relatedmacular degeneration such as wet AMD or dry AMD), Toxoplasmosis,Choroidal Melanoma, Acquired Retinoschisis, Hollenhorst Plaque,Idiopathic Central Serous Chorioretinopathy, Macular Hole, PresumedOcular Histoplasmosis Syndrome, Retinal Macroaneursym, RetinitisPigmentosa, Retinal Detachment, Hypertensive Retinopathy, RetinalPigment Epithelium (RPE) Detachment, Papillophlebitis, Ocular IschemicSyndrome, Coats' Disease, Leber's Miliary Aneurysm, ConjunctivalNeoplasms, Allergic Conjunctivitis, Vernal Conjunctivitis, AcuteBacterial Conjunctivitis, Allergic Conjunctivitis & VernalKeratoconjunctivitis, Viral Conjunctivitis, Bacterial Conjunctivitis,Chlamydial & Gonococcal Conjunctivitis, Conjunctival Laceration,Episcleritis, Scleritis, Pingueculitis, Pterygium, Superior LimbicKeratoconjunctivitis (SLK of Theodore), Toxic Conjunctivitis,Conjunctivitis with Pseudomembrane, Giant Papillary Conjunctivitis,Terrien's Marginal Degeneration, Acanthamoeba Keratitis, FungalKeratitis, Filamentary Keratitis, Bacterial Keratitis, KeratitisSicca/Dry Eye Syndrome, Bacterial Keratitis, Herpes Simplex Keratitis,Sterile Corneal Infiltrates, Phlyctenulosis, Corneal Abrasion &Recurrent Corneal Erosion, Corneal Foreign Body, Chemical Burs,Epithelial Basement Membrane Dystrophy (EBMD), Thygeson's SuperficialPunctate Keratopathy, Corneal Laceration, Salzmann's NodularDegeneration, Fuchs' Endothelial Dystrophy, Crystalline LensSubluxation, Ciliary-Block Glaucoma, Primary Open-Angle Glaucoma,Pigment Dispersion Syndrome and Pigmentary Glaucoma, PseudoexfoliationSyndrom and Pseudoexfoliative Glaucoma, Anterior Uveitis, Primary OpenAngle Glaucoma, Uveitic Glaucoma & Glaucomatocyclitic Crisis, PigmentDispersion Syndrome & Pigmentary Glaucoma, Acute Angle Closure Glaucoma,Anterior Uveitis, Hyphema, Angle Recession Glaucoma, Lens InducedGlaucoma, Pseudoexfoliation Syndrome and Pseudoexfoliative Glaucoma,Axenfeld-Rieger Syndrome, Neovascular Glaucoma, Pars Planitis, ChoroidalRupture, Duane's Retraction Syndrome, Toxic/Nutritional OpticNeuropathy, Aberrant Regeneration of Cranial Nerve III, IntracranialMass Lesions, Carotid-Cavernous Sinus Fistula, Anterior Ischemic OpticNeuropathy, Optic Disc Edema & Papilledema, Cranial Nerve III Palsy,Cranial Nerve IV Palsy, Cranial Nerve VI Palsy, Cranial Nerve VII(Facial Nerve) Palsy, Horner's Syndrome, Internuclear Opthalmoplegia,Optic Nerve Head Hypoplasia, Optic Pit, Tonic Pupil, Optic Nerve HeadDrusen, Demyelinating Optic Neuropathy (Optic Neuritis, RetrobulbarOptic Neuritis), Amaurosis Fugax and Transient Ischemic Attack,Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum,Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis,Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal CellCarcinoma, Herpes Zoster Ophthalmicus, Pediculosis & Phthiriasis,Blow-out Fracture, Chronic Epiphora, Dacryocystitis, Herpes SimplexBlepharitis, Orbital Cellulitis, Senile Entropion, and Squamous CellCarcinoma.

By “cardiovascular disease” is meant and disease or condition affectingthe heart and vasculature, including but not limited to, coronary heartdisease (CHD), cerebrovascular disease (CVD), aortic stenosis,peripheral vascular disease, atherosclerosis, arteriosclerosis,myocardial infarction (heart attack), cerebrovascular diseases (stroke),transient ischaemic attacks (TIA), angina (stable and unstable), atrialfibrillation, arrhythmia, valvular disease, and/or congestive heartfailure.

In one embodiment of the present invention, each sequence of an siNAmolecule of the invention is independently about 18 to about 24nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22,23, or 24 nucleotides in length. In another embodiment, the siNAduplexes of the invention independently comprise about 17 to about 23base pairs (e.g., about 17, 18, 19, 20, 21, 22, or 23). In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38,39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 16to about 22 (e.g., about 16, 17, 18, 19, 20, 21 or 22) base pairs.Exemplary siNA molecules of the invention are shown in Table II.Exemplary synthetic siNA molecules of the invention are shown in TableIII and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through direct dermal application, transdermal application, orinjection, with or without their incorporation in biopolymers. Inparticular embodiments, the nucleic acid molecules of the inventioncomprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples ofsuch nucleic acid molecules consist essentially of sequences defined inthese tables and figures. Furthermore, the chemically modifiedconstructs described in Table IV can be applied to any siNA sequence ofthe invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribofuranose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to forpreventing or treating cancers and other proliferative conditions, viralinfection, inflammatory disease, autoimmunity, respiratory disease,pulmonary disease, cardiovascular disease, neurological disease, renaldisease, ocular disease, liver disease, mitochondrial disease, endocrinedisease, prion disease, or reproduction related diseases and conditionsin a subject or organism. In one embodiment, siNA molecules of theinvention are used in combination with anti-inflammatory agents orbronchodilators as are known in the art to treat or prevent inflammatoryand respiratory diseases and/or conditions in a subject or organism.

For example, the siNA molecules can be administered to a subject or canbe administered to other appropriate cells evident to those skilled inthe art, individually or in combination with one or more drugs (e.g.,statins, hypertensive agents etc.) under conditions suitable for thetreatment.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of an siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms an siNA molecule. Non-limitingexamples of such expression vectors are described in Paul et al., 2002,Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, NatureBiotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500;and Novina et al., 2002, Nature Medicine, advance online publicationdoi: 10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for an siNA molecule having complementarity to aRNA molecule referred to by a Genbank Accession numbers, for exampleGenbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form ansiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs. Theantisense strand of constructs A-F comprise sequence complementary toany target nucleic acid sequence of the invention. Furthermore, when aglyceryl moiety (L) is present at the 3′-end of the antisense strand forany construct shown in FIG. 4 A-F, the modified internucleotide linkageis optional.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all nucleotidespresent are ribonucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target RNA sequence, and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. A modified internucleotide linkage, such as aphosphorothioate, phosphorodithioate or other modified internucleotidelinkage as described herein, shown as “s”, optionally connects the (N N)nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides,optionally having a 3′-terminal glyceryl moiety and wherein the twoterminal 3′-nucleotides are optionally complementary to the target RNAsequence, and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,and having one 3′-terminal phosphorothioate internucleotide linkage andwherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-deoxy nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 5A-F shows non-limiting examples of specific chemically-modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to an IL-4R siNA sequence. Such chemicalmodifications can be applied to any interleukin and/or interleukinreceptor sequence and/or interleukin and/or interleukin receptorpolymorphism sequence.

FIG. 6 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined interleukin and/or interleukin receptor targetsequence, wherein the sense region comprises, for example, about 19, 20,21, or 22 nucleotides (N) in length, which is followed by a loopsequence of defined sequence (X), comprising, for example, about 3 toabout 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence thatwill result in an siNA transcript having specificity for a interleukinand/or interleukin receptor target sequence and havingself-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) tolinearize the sequence, thus allowing extension of a complementarysecond DNA strand using a primer to the 3′-restriction sequence of thefirst strand. The double-stranded DNA is then inserted into anappropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double-stranded siNAconstructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) sitesequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined interleukin and/or interleukin receptor targetsequence, wherein the sense region comprises, for example, about 19, 20,21, or 22 nucleotides (N) in length, and which is followed by a3′-restriction site (R2) which is adjacent to a loop sequence of definedsequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific toR1 and R2 to generate a double-stranded DNA which is then inserted intoan appropriate vector for expression in cells. The transcriptioncassette is designed such that a U6 promoter region flanks each side ofthe dsDNA which generates the separate sense and antisense strands ofthe siNA. Poly T termination sequences can be added to the constructs togenerate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein theantisense region of the siNA constructs has complementarity to targetsites across the target nucleic acid sequence, and wherein the senseregion comprises sequence complementary to the antisense region of thesiNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted intovectors such that (FIG. 9C) transfection of a vector into cells resultsin the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associatedwith modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced toidentify efficacious target sites within the target nucleic acidsequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g. humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design selfcomplementary DFO constructs utilizing palindrome and/or repeat nucleicacid sequences that are identified in a target nucleic acid sequence.(i) A palindrome or repeat sequence is identified in a nucleic acidtarget sequence. (ii) A sequence is designed that is complementary tothe target nucleic acid sequence and the palindrome sequence. (iii) Aninverse repeat sequence of the non-palindrome/repeat portion of thecomplementary sequence is appended to the 3′-end of the complementarysequence to generate a self complementary DFO molecule comprisingsequence complementary to the nucleic acid target. (iv) The DFO moleculecan self-assemble to form a double-stranded oligonucleotide. FIG. 14Bshows a non-limiting representative example of a duplex formingoligonucleotide sequence. FIG. 14C shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence. FIG. 14D shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence followed by interaction with a target nucleicacid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementaryDFO constructs utilizing palindrome and/or repeat nucleic acid sequencesthat are incorporated into the DFO constructs that have sequencecomplementary to any target nucleic acid sequence of interest.Incorporation of these palindrome/repeat sequences allow the design ofDFO constructs that form duplexes in which each strand is capable ofmediating modulation of target gene expression, for example by RNAi.First, the target sequence is identified. A complementary sequence isthen generated in which nucleotide or non-nucleotide modifications(shown as X or Y) are introduced into the complementary sequence thatgenerate an artificial palindrome (shown as XYXYXY in the Figure). Aninverse repeat of the non-palindrome/repeat complementary sequence isappended to the 3′-end of the complementary sequence to generate a selfcomplementary DFO comprising sequence complementary to the nucleic acidtarget. The DFO can self-assemble to form a double-strandedoligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. FIG. 16A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 16B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first and second complementaryregions are situated at the 5′-ends of each polynucleotide sequence inthe multifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. FIG. 17A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 17B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first complementary region issituated at the 5′-end of the polynucleotide sequence in themultifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. In one embodiment,these multifunctional siNA constructs are processed in vivo or in vitroto generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifunctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. FIG. 18Ashows a non-limiting example of a multifunctional siNA molecule having afirst region that is complementary to a first target nucleic acidsequence (complementary region 1) and a second region that iscomplementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 3′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 18B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 5′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifunctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. FIG. 19A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 19B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins,for example a cytokine and its corresponding receptor, differing viralstrains, a virus and a cellular protein involved in viral infection orreplication, or differing proteins involved in a common or divergentbiologic pathway that is implicated in the maintenance of progression ofdisease. Each strand of the multifunctional siNA construct comprises aregion having complementarity to separate target nucleic acid molecules.The multifunctional siNA molecule is designed such that each strand ofthe siNA can be utilized by the RISC complex to initiate RNAinterference mediated cleavage of its corresponding target. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarityto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC complex to initiate RNA interferencemediated cleavage of its corresponding target region. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 22 shows a non-limiting example of reduction of IL-4R mRNA in HeLacells mediated by siNAs that target IL-4R mRNA. HeLa cells weretransfected with 0.25 ug/well of lipid complexed with 25 nM siNA. ActivesiNA constructs comprising Stab 9/22 stabilization chemistry werecompared to matched chemistry irrelevant siNA control constructs (IC),and cells transfected with lipid alone (transfection control). As shownin the figure, the siNA constructs significantly reduce IL-4R RNAexpression.

DETAILED DESCRIPTION OF THE INVENTION

Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically-modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or an siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing an siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of an siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table V outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-benzodithiol-3-one1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table V outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA·3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO:1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA·3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

An siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to an siNA molecule of the invention or thesense and antisense strands of an siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatments by affording the possibility of combination therapies(e.g., multiple siNA molecules targeted to different genes; nucleic acidmolecules coupled with known small molecule modulators; or intermittenttreatment with combinations of molecules, including different motifsand/or other chemical or biological molecules). The treatment ofsubjects with siNA molecules can also include combinations of differenttypes of nucleic acid molecules, such as enzymatic nucleic acidmolecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate,decoys, and aptamers.

In another aspect an siNA molecule of the invention comprises one ormore 5′ and/or a 3′-cap structure, for example on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and suitable heterocyclic groups include furanyl, thienyl, pyridyl,pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl andthe like, all optionally substituted. An “amide” refers to an—C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An“ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylarylor hydrogen.

“Nucleotide” as used herein, and as recognized in the art, includesnatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2 ,4, 6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1 carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′—NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

An siNA molecule of the invention can be adapted for use to prevent ortreat cancers and other proliferative conditions, viral infection,inflammatory disease, autoimmunity, respiratory disease, pulmonarydisease, cardiovascular disease, neurological disease, renal disease,ocular disease, liver disease, mitochondrial disease, endocrine disease,prion disease, reproduction related diseases and conditions, and/or anyother trait, disease or condition that is related to or will respond tothe levels of interleukin and/or interleukin receptor in a cell ortissue, alone or in combination with other therapies. For example, ansiNA molecule can comprise a delivery vehicle, including liposomes, foradministration to a subject, carriers and diluents and their salts,and/or can be present in pharmaceutically acceptable formulations.Methods for the delivery of nucleic acid molecules are described inAkhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies forAntisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al.,1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb.Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser.,752, 184-192, all of which are incorporated herein by reference.Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO94/02595 further describe the general methods for delivery of nucleicacid molecules. These protocols can be utilized for the delivery ofvirtually any nucleic acid molecule. Nucleic acid molecules can beadministered to cells by a variety of methods known to those of skill inthe art, including, but not restricted to, encapsulation in liposomes,by iontophoresis, or by incorporation into other vehicles, such asbiodegradable polymers, hydrogels, cyclodextrins (see for exampleGonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al.,International PCT publication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In another embodiment,the nucleic acid molecules of the invention can also be formulated orcomplexed with polyethyleneimine and derivatives thereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives.

In one embodiment, an siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, an siNA molecule of the invention is complexed withdelivery systems as described in U.S. Patent Application Publication No.2003077829 and International PCT Publication Nos. WO 00/03683 and WO02/087541, all incorporated by reference herein in their entiretyincluding the drawings.

In addition, the invention features the use of methods to deliver thenucleic acid molecules of the instant invention to the central nervoussystem and/or peripheral nervous system. Experiments have demonstratedthe efficient in vivo uptake of nucleic acids by neurons. As an exampleof local administration of nucleic acids to nerve cells, Sommer et al.,1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe a study in which a15mer phosphorothioate antisense nucleic acid molecule to c-fos isadministered to rats via microinjection into the brain. Antisensemolecules labeled with tetramethylrhodamine-isothiocyanate (TRITC) orfluorescein isothiocyanate (FITC) were taken up by exclusively byneurons thirty minutes post-injection. A diffuse cytoplasmic stainingand nuclear staining was observed in these cells. As an example ofsystemic administration of nucleic acid to nerve cells, Epa et al.,2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mousestudy in which beta-cyclodextrin-adamantane-oligonucleotide conjugateswere used to target the p75 neurotrophin receptor in neuronallydifferentiated PC12 cells. Following a two week course of IPadministration, pronounced uptake of p75 neurotrophin receptor antisensewas observed in dorsal root ganglion (DRG) cells. In addition, a markedand consistent down-regulation of p75 was observed in DRG neurons.Additional approaches to the targeting of nucleic acid to neurons aredescribed in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle etal., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, BrainResearch, 784(1, 2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199;Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, BrainRes. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39.Nucleic acid molecules of the invention are therefore amenable todelivery to and uptake by cells that express repeat expansion allelicvariants for modulation of RE gene expression. The delivery of nucleicacid molecules of the invention, targeting RE is provided by a varietyof different strategies. Traditional approaches to CNS delivery that canbe used include, but are not limited to, intrathecal andintracerebroventricular administration, implantation of catheters andpumps, direct injection or perfusion at the site of injury or lesion,injection into the brain arterial system, or by chemical or osmoticopening of the blood-brain barrier. Other approaches can include the useof various transport and carrier systems, for example though the use ofconjugates and biodegradable polymers. Furthermore, gene therapyapproaches, for example as described in Kaplitt et al., U.S. Pat. No.6,180,613 and Davidson, WO 04/013280, can be used to express nucleicacid molecules in the CNS.

In addition, the invention features the use of methods to deliver thenucleic acid molecules of the instant invention to hematopoietic cells,including monocytes and lymphocytes. These methods are described indetail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2),920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion andPhillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei,1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, NucleicAcids Research, 22(22), 4681-8. Such methods, as described above,include the use of free oligonucleotide, cationic lipid formulations,liposome formulations including pH sensitive liposomes andimmunoliposomes, and bioconjugates including oligonucleotides conjugatedto fusogenic peptides, for the transfection of hematopoietic cells witholigonucleotides.

In one embodiment, a compound, molecule, or composition for thetreatment of ocular conditions (e.g., macular degeneration, diabeticretinopathy etc.) is administered to a subject intraocularly or byintraocular means. In another embodiment, a compound, molecule, orcomposition for the treatment of ocular conditions (e.g., maculardegeneration, diabetic retinopathy etc.) is administered to a subjectperiocularly or by periocular means (see for example Ahlheim et al.,International PCT publication No. WO 03/24420). In one embodiment, ansiNA molecule and/or formulation or composition thereof is administeredto a subject intraocularly or by intraocular means. In anotherembodiment, an siNA molecule and/or formulation or composition thereofis administered to a subject periocularly or by periocular means.Periocular administration generally provides a less invasive approach toadministering siNA molecules and formulation or composition thereof to asubject (see for example Ahlheim et al., International PCT publicationNo. WO 03/24420). The use of periocular administration also minimizesthe risk of retinal detachment, allows for more frequent dosing oradministration, provides a clinically relevant route of administrationfor macular degeneration and other optic conditions, and also providesthe possibility of using reservoirs (e.g., implants, pumps or otherdevices) for drug delivery.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered directly or topically (e.g.,locally) to the dermis or follicles as is generally known in the art(see for example Brand, 2001, Curr. Opin. Mol. Ther., 3, 244-8; Regnieret al., 1998, J. Drug Target, 5, 275-89; Kanikkannan, 2002, BioDrugs,16, 339-47; Wraight et al., 2001, Pharmacol. Ther., 90, 89-104; andPreat and Dujardin, 2001, STP PharmaSciences, 11, 57-68.

In one embodiment, dermal delivery systems of the invention include, forexample, aqueous and nonaqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and nonaqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inone embodiment, the pharmaceutically acceptable carrier is a liposome ora transdermal enhancer. Examples of liposomes which can be used in thisinvention include the following: (1) CellFectin, 1:1.5 (M/M) liposomeformulation of the cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine anddioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

In one embodiment, transmucosal delivery systems of the inventioninclude patches, tablets, suppositories, pessaries, gels and creams, andcan contain excipients such as solubilizers and enhancers (e.g.,propylene glycol, bile salts and amino acids), and other vehicles (e.g.,polyethylene glycol, fatty acid esters and derivatives, and hydrophilicpolymers such as hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, nucleic acid molecules of the invention areadministered to the central nervous system (CNS) or peripheral nervoussystem (PNS). Experiments have demonstrated the efficient in vivo uptakeof nucleic acids by neurons. As an example of local administration ofnucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. AcidDrug Dev., 8, 75, describe a study in which a 15mer phosphorothioateantisense nucleic acid molecule to c-fos is administered to rats viamicroinjection into the brain. Antisense molecules labeled withtetramethylrhodamine-isothiocyanate (TRITC) or fluoresceinisothiocyanate (FITC) were taken up by exclusively by neurons thirtyminutes post-injection. A diffuse cytoplasmic staining and nuclearstaining was observed in these cells. As an example of systemicadministration of nucleic acid to nerve cells, Epa et al., 2000,Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse studyin which beta-cyclodextrin-adamantane-oligonucleotide conjugates wereused to target the p75 neurotrophin receptor in neuronallydifferentiated PC12 cells. Following a two week course of IPadministration, pronounced uptake of p75 neurotrophin receptor antisensewas observed in dorsal root ganglion (DRG) cells. In addition, a markedand consistent down-regulation of p75 was observed in DRG neurons.Additional approaches to the targeting of nucleic acid to neurons aredescribed in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle etal., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, BrainResearch, 784(1, 2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199;Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, BrainRes. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39.Nucleic acid molecules of the invention are therefore amenable todelivery to and uptake by cells in the CNS and/or PNS.

The delivery of nucleic acid molecules of the invention to the CNS isprovided by a variety of different strategies. Traditional approaches toCNS delivery that can be used include, but are not limited to,intrathecal and intracerebroventricular administration, implantation ofcatheters and pumps, direct injection or perfusion at the site of injuryor lesion, injection into the brain arterial system, or by chemical orosmotic opening of the blood-brain barrier. Other approaches can includethe use of various transport and carrier systems, for example though theuse of conjugates and biodegradable polymers. Furthermore, gene therapyapproaches, for example as described in Kaplitt et al., U.S. Pat. No.6,180,613 and Davidson, WO 04/013280, can be used to express nucleicacid molecules in the CNS.

In one embodiment, the nucleic acid molecules of the invention areadministered via pulmonary delivery, such as by inhalation of an aerosolor spray dried formulation administered by an inhalation device ornebulizer, providing rapid local uptake of the nucleic acid moleculesinto relevant pulmonary tissues. Solid particulate compositionscontaining respirable dry particles of micronized nucleic acidcompositions can be prepared by grinding dried or lyophilized nucleicacid compositions, and then passing the micronized composition through,for example, a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprising the nucleicacid compositions of the invention can optionally contain a dispersantwhich serves to facilitate the formation of an aerosol as well as othertherapeutic compounds. A suitable dispersant is lactose, which can beblended with the nucleic acid compound in any suitable ratio, such as a1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition ofthe invention can be produced by any suitable means, such as with anebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers arecommercially available devices which transform solutions or suspensionsof an active ingredient into a therapeutic aerosol mist either by meansof acceleration of a compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers comprise the active ingredient in aliquid carrier in an amount of up to 40% w/w preferably less than 20%w/w of the formulation. The carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, for example, sodium chloride or other suitable salts.Optional additives include preservatives if the formulation is notprepared sterile, for example, methyl hydroxybenzoate, anti-oxidants,flavorings, volatile oils, buffering agents and emulsifiers and otherformulation surfactants. The aerosols of solid particles comprising theactive composition and surfactant can likewise be produced with anysolid particulate aerosol generator. Aerosol generators foradministering solid particulate therapeutics to a subject produceparticles which are respirable, as explained above, and generate avolume of aerosol containing a predetermined metered dose of atherapeutic composition at a rate suitable for human administration. Oneillustrative type of solid particulate aerosol generator is aninsufflator. Suitable formulations for administration by insufflationinclude finely comminuted powders which can be delivered by means of aninsufflator. In the insufflator, the powder, e.g., a metered dosethereof effective to carry out the treatments described herein, iscontained in capsules or cartridges, typically made of gelatin orplastic, which are either pierced or opened in situ and the powderdelivered by air drawn through the device upon inhalation or by means ofa manually-operated pump. The powder employed in the insufflatorconsists either solely of the active ingredient or of a powder blendcomprising the active ingredient, a suitable powder diluent, such aslactose, and an optional surfactant. The active ingredient typicallycomprises from 0.1 to 100 w/w of the formulation. A second type ofillustrative aerosol generator comprises a metered dose inhaler. Metereddose inhalers are pressurized aerosol dispensers, typically containing asuspension or solution formulation of the active ingredient in aliquefied propellant. During use these devices discharge the formulationthrough a valve adapted to deliver a metered volume to produce a fineparticle spray containing the active ingredient. Suitable propellantsinclude certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation canadditionally contain one or more co-solvents, for example, ethanol,emulsifiers and other formulation surfactants, such as oleic acid orsorbitan trioleate, anti-oxidants and suitable flavoring agents. Othermethods for pulmonary delivery are described in, for example US PatentApplication No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728;6,565,885.

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PharmaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, an siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. Nos. 6,528,631; 6,335,434; 6,235,886; 6,153,737; 5,214,136;5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedinto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as tablets, capsules orelixirs for oral administration, suppositories for rectaladministration, sterile solutions, suspensions for injectableadministration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, transdermal, or by injection. Such forms should notprevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes that lead to systemicabsorption include, without limitation: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.Each of these administration routes exposes the siNA molecules of theinvention to an accessible diseased tissue. The rate of entry of a druginto the circulation has been shown to be a function of molecular weightor size. The use of a liposome or other drug carrier comprising thecompounds of the instant invention can potentially localize the drug,for example, in certain tissue types, such as the tissues of thereticular endothelial system (RES). A liposome formulation that canfacilitate the association of drug with the surface of cells, such as,lymphocytes and macrophages is also useful. This approach can provideenhanced delivery of the drug to target cells by taking advantage of thespecificity of macrophage and lymphocyte immune recognition of abnormalcells, such as cancer cells.

By “pharmaceutically acceptable formulation” is meant a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as Pluronic P85),which can enhance entry of drugs into the CNS (Jolliet-Riant andTillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery after intracerebral implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge,Mass.); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate, which can deliver drugs across the blood brainbarrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBSLett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596;Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada etal., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999,PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatennary or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 10/151,116, filed May 17, 2002. In one embodiment, nucleic acidmolecules of the invention are complexed with or covalently attached tonanoparticles, such as Hepatitis B virus S, M, or L evelope proteins(see for example Yamado et al., 2003, Nature Biotechnology, 21, 885). Inone embodiment, nucleic acid molecules of the invention are deliveredwith specificity for human tumor cells, specifically non-apoptotic humantumor cells including for example T-cells, hepatocytes, breast carcinomacells, ovarian carcinoma cells, melanoma cells, intestinal epithelialcells, prostate cells, testicular cells, non-small cell lung cancers,small cell lung cancers, etc.

In one embodiment, an siNA molecule of the invention is designed orformulated to specifically target endothelial cells or tumor cells. Forexample, various formulations and conjugates can be utilized tospecifically target endothelial cells or tumor cells, includingPEI-PEG-folate, PEI-PEG-RGD, PEI-PEG-biotin, PEI-PEG-cholesterol, andother conjugates known in the art that enable specific targeting toendothelial cells and/or tumor cells.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; propulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe etal., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,45). Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856).

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pats.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from a subject followed by reintroduction into thesubject, or by any other means that would allow for introduction intothe desired target cell (for a review see Couture et al., 1996, TIG.,12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of ansiNA duplex, or a single self-complementary strand that self hybridizesinto an siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi: 10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. U S A, 87, 6743-7;Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al.,1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol.,10, 4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U. S. A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of an siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of an siNA molecule, wherein the sequenceis operably linked to the 3′-end of the open reading frame and whereinthe sequence is operably linked to the initiation region, the intron,the open reading frame and the termination region in a manner whichallows expression and/or delivery of the siNA molecule.

Interleukin Biology and Biochemistry

The following discussion is adapted from R&D Systems Mini-Reviews andTech Notes, Cytokine Mini-Reviews, Copyright©2002 R&D Systems.Interleukin 2 (IL-2) is a lymphokine synthesized and secreted primarilyby T helper lymphocytes that have been activated by stimulation withcertain mitogens or by interaction of the T cell receptor complex withantigen/MHC complexes on the surfaces of antigen-presenting cells. Theresponse of T helper cells to activation is induction of the expressionof IL-2 and receptors for IL-2 and, subsequently, clonal expansion ofantigen-specific T cells. At this level IL-2 is an autocrine factor,driving the expansion of the antigen-specific cells. IL-2 also acts as aparacrine factor, influencing the activity of other cells, both withinthe immune system and outside of it. B cells and natural killer (NK)cells respond, when properly activated, to IL-2. The so-calledlymphocyte activated killer, or LAK cells, appear to be derived from NKcells under the influence of IL-2.

The biological activities of IL-2 are mediated through the binding ofIL-2 to a multisubunit cellular receptor. Although three distincttransmembrane glycoprotein subunits contribute to the formation of thehigh affinity IL-2 receptor, various combinations of receptor subunits(alpha, beta, gamma) are known to occur.

Interleukin 1 (IL-1) is a general name for two distinct proteins, IL-1aand IL-1b, that are considered the first of a family of regulatory andinflammatory cytokines. Along with IL-1 receptor antagonist (IL-1ra)2and IL-18,3 these molecules play important roles in the up- anddown-regulation of acute inflammation. In the immune system, theproduction of IL-1 is typically induced, generally resulting ininflammation. IL-1b and TNF-a are generally thought of as prototypicalpro-inflammatory cytokines. The effects of IL-1, however, are notlimited to inflammation, as IL-1 has also been associated with boneformation and remodeling, insulin secretion, appetite regulation, feverinduction, neuronal phenotype development, and IGF/GH physiology. IL-1has also been known by a number of alternative names, includinglymphocyte activating factor, endogenous pyrogen, catabolin,hemopoietin-1, melanoma growth inhibition factor, and osteoclastactivating factor. IL-1a and IL-1b exert their effects by binding tospecific receptors. Two distinct IL-1 receptor binding proteins, plus anon-binding signaling accessory protein have been identified to date.Each have three extracellular immunoglobulin-like (Ig-like) domains,qualifying them for membership in the type IV cytokine receptor family.

Interleukin-4 (IL-4) mediates important pro-inflammatory functions inasthma including induction of the IgE isotype switch, expression ofvascular cell adhesion molecule-1 (VCAM-1), promotion of eosinophiltransmigration across endothelium, mucus secretion, and differentiationof T helper type 2 lymphocytes leading to cytokine release. Asthma hasbeen linked to polymorphisms in the IL-4 gene promoter and proteinsinvolved in IL-4 signaling. Soluble recombinant IL-4 receptor lackstransmembrane and cytoplasmic activating domains and can thereforesequester IL-4 without mediating cellular activation. Genetic variantswithin the IL-4 signaling pathway might contribute to the risk ofdeveloping asthma in a given individual. A number of polymorphisms havebeen described within the IL-4 receptor a (IL-4Rα) gene, and inaddition, polymorphism occurs in the promoter for the IL-4 gene itself(see for example Hall, 2000, Respir. Res., 1, 6-8 and Ober et al., 2000,Am J Hum Genet., 66, 517-526, for a review). The type 2 cytokine IL-13,which shares a receptor component and signaling pathways with IL-4, wasfound to be necessary and sufficient for the expression of allergicasthma (see Wills-Karp et al., 1998, Science, 282, 2258-61). IL-13induces the pathophysiological features of asthma in a manner that isindependent of immunoglobulin E and eosinophils. Thus, IL-13 is criticalto allergen-induced asthma but operates through mechanisms other thanthose that are classically implicated in allergic responses.

Human IL-5 is a 134 amino acid polypeptide with a predicted mass of 12.5kDa. It is secreted by a restricted number of mesenchymal cell types. Inits native state, mature IL-5 is synthesized as a 115 aa, highlyglycosylated 22 kDa monomer that forms a 40-50 kDa disulfide-linkedhomodimer. Although the content of carbohydrate is high, carbohydrate isnot needed for bioactivity. Monomeric IL-5 has no activity; a homodimeris required for function. This is in contrast to the receptor-relatedcytokines IL-3 and GM-CSF, which exist only as monomers. Just as oneIL-3 and GM-CSF monomer binds to one receptor, one IL-5 homodimer isable to engage only one IL-5 receptor. It has been suggested that IL-5(as a dimer) undergoes a general conformational change after binding toone receptor molecule, and this change precludes binding to a secondreceptor. The receptor for IL-5 consists of a ligand binding a-subunitand a non-ligand binding (common) signal transducing b-subunit that isshared by the receptors for IL-3 and GM-CSF. IL-5 appears to perform anumber of functions on eosinophils. These include the down modulation ofMac-1, the upregulation of receptors for IgA and IgG, the stimulation oflipid mediator (leukotriene C4 and PAF) secretion and the induction ofgranule release. IL-5 also promotes the growth and differentiation ofeosinophils.

Interleukin 6 (IL-6) is considered a prototypic pleiotrophic cytokine.This is reflected in the variety of names originally assigned to IL-6based on function, including Interferon b2, IL-1-inducible 26 kDProtein, Hepatocyte Stimulating Factor, Cytotoxic T-cell DifferentiationFactor, B cell Differentiation Factor (BCDF) and/or B cell StimulatoryFactor 2 (BSF2). A number of cytokines make up an IL-6 cytokine family.Membership in this family is typically based on a helical cytokinestructure and receptor subunit makeup. The functional receptor for IL-6is a complex of two transmembrane glycoproteins (gp130 and IL-6receptor) that are members of the Class I cytokine receptor superfamily.

Because of the central role of the interleukin family of cytokines inthe mediation of immune and inflammatory responses, modulation ofinterleukin expression and/or activity can provide important functionsin therapeutic and diagnostic applications. The use of small interferingnucleic acid molecules targeting interleukins and their correspondingreceptors therefore provides a class of novel therapeutic agents thatcan be used in the treatment of cancers, proliferative diseases,inflammatory disease, respiratory disease, pulmonary disease,cardiovascular disease, autoimmune disease, infectious disease, priordisease, renal disease, transplant rejection, or any other disease orcondition that responds to modulation of interleukin and interleukinreceptor genes.

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of an siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H2O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H20 followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 3 Selection of siNA Molecule Target Fites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragmentsor subsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

2. In some instances the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence; the goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.

3. In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided whenever better sequences are available. CCC is searched in thetarget strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

8. Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex (see Tables II and III). Ifterminal TT residues are desired for the sequence (as described inparagraph 7), then the two 3′ terminal nucleotides of both the sense andantisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture oranimal model system to identify the most active siNA molecule or themost preferred target site within the target RNA sequence.

10. Other design considerations can be used when selecting targetnucleic acid sequences, see for example Reynolds et al., 2004, NatureBiotechnology Advanced Online Publication, 1 Feb. 2004,doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32,doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to ainterleukin and/or interleukin receptor target sequence is used toscreen for target sites in cells expressing interleukin and/orinterleukin receptor RNA, such as cultured Jurkat, HeLa, or 293T cells.The general strategy used in this approach is shown in FIG. 9. Anon-limiting example of such is a pool comprising sequences having anyof SEQ ID NOS1-1828. Cells expressing interleukin and/or interleukinreceptor (e.g., Jurkat, HeLa, or 293T cells) are transfected with thepool of siNA constructs and cells that demonstrate a phenotypeassociated with interleukin and/or interleukin receptor inhibition aresorted. The pool of siNA constructs can be expressed from transcriptioncassettes inserted into appropriate vectors (see for example FIG. 7 andFIG. 8). The siNA from cells demonstrating a positive phenotypic change(e.g., decreased interleukin and/or interleukin receptor mRNA levels ordecreased interleukin and/or interleukin receptor protein expression),are sequenced to determine the most suitable target site(s) within thetarget interleukin and/or interleukin receptor RNA sequence.

Example 4 Interleukin and Interleukin Receptor Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the interleukinand/or interleukin receptor RNA target and optionally prioritizing thetarget sites on the basis of folding (structure of any given sequenceanalyzed to determine siNA accessibility to the target), by using alibrary of siNA molecules as described in Example 3, or alternately byusing an in vitro siNA system as described in Example 6 herein. siNAmolecules were designed that could bind each target and are optionallyindividually analyzed by computer folding to assess whether the siNAmolecule can interact with the target sequence. Varying the length ofthe siNA molecules can be chosen to optimize activity. Generally, asufficient number of complementary nucleotide bases are chosen to bindto, or otherwise interact with, the target RNA, but the degree ofcomplementarity can be modulated to accommodate siNA duplexes or varyinglength or base composition. By using such methodologies, siNA moleculescan be designed to target sites within any known RNA sequence, forexample those RNA sequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Usman et al., U.S. Pat. Nos. 5,831,071,6,353,098, 6,437,117, and Bellon et al., U.S. Pat. Nos. 6,054,576,6,162,909, 6,303,773, or Scaringe supra, incorporated by referenceherein in their entireties. Additionally, deprotection conditions can bemodified to provide the best possible yield and purity of siNAconstructs. For example, applicant has observed that oligonucleotidescomprising 2′-deoxy-2′-fluoro nucleotides can degrade underinappropriate deprotection conditions. Such oligonucleotides aredeprotected using aqueous methylamine at about 35° C. for 30 minutes. Ifthe 2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting interleukin and/or interleukinreceptor RNA targets. The assay comprises the system described by Tuschlet al., 1999, Genes and Development, 13, 3191-3197 and Zamore et al.,2000, Cell, 101, 25-33 adapted for use with interleukin and/orinterleukin receptor target RNA. A Drosophila extract derived fromsyncytial blastoderm is used to reconstitute RNAi activity in vitro.Target RNA is generated via in vitro transcription from an appropriateinterleukin and/or interleukin receptor expressing plasmid using T7 RNApolymerase or via chemical synthesis as described herein. Sense andantisense siNA strands (for example 20 uM each) are annealed byincubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH,pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C. followed by 1hour at 37° C., then diluted in lysis buffer (for example 100 mMpotassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate).Annealing can be monitored by gel electrophoresis on an agarose gel inTBE buffer and stained with ethidium bromide. The Drosophila lysate isprepared using zero to two-hour-old embryos from Oregon R fliescollected on yeasted molasses agar that are dechorionated and lysed. Thelysate is centrifuged and the supernatant isolated. The assay comprisesa reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM finalconcentration), and 10% [vol/vol] lysis buffer containing siNA (10 nMfinal concentration). The reaction mixture also contains 10 mM creatinephosphate, 10 ug/ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM ofeach amino acid. The final concentration of potassium acetate isadjusted to 100 mM. The reactions are pre-assembled on ice andpreincubated at 25° C. for 10 minutes before adding RNA, then incubatedat 25° C. for an additional 60 minutes. Reactions are quenched with 4volumes of 1.25× Passive Lysis Buffer (Promega). Target RNA cleavage isassayed by RT-PCR analysis or other methods known in the art and arecompared to control reactions in which siNA is omitted from thereaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²P] CTP, passed over aG 50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without siNA and the cleavage productsgenerated by the assay.

In one embodiment, this assay is used to determine target sites theinterleukin and/or interleukin receptor RNA target for siNA mediatedRNAi cleavage, wherein a plurality of siNA constructs are screened forRNAi mediated cleavage of the interleukin and/or interleukin receptorRNA target, for example, by analyzing the assay reaction byelectrophoresis of labeled target RNA, or by Northern blotting, as wellas by other methodology well known in the art.

Example 7 Nucleic Acid Inhibition of Interleukin and InterleukinReceptor Target RNA in Vitro

siNA molecules targeted to the human interleukin and/or interleukinreceptor RNA are designed and synthesized as described above. Thesenucleic acid molecules can be tested for cleavage activity in vivo, forexample, using the following procedure. The target sequences and thenucleotide location within the interleukin and/or interleukin receptorRNA are given in Table II and III.

Two formats are used to test the efficacy of siNAs targeting interleukinand/or interleukin receptor. First, the reagents are tested in cellculture using, for example, Jurkat, HeLa, or 293T cells, to determinethe extent of RNA and protein inhibition. siNA reagents (e.g.; seeTables II and III) are selected against the interleukin and/orinterleukin receptor target as described herein. RNA inhibition ismeasured after delivery of these reagents by a suitable transfectionagent to, for example, cultured Jurkat, HeLa, or 293T cells. Relativeamounts of target RNA are measured versus actin using real-time PCRmonitoring of amplification (e.g., ABI 7700 TAQMAN®). A comparison ismade to a mixture of oligonucleotide sequences made to unrelated targetsor to a randomized siNA control with the same overall length andchemistry, but randomly substituted at each position. Primary andsecondary lead reagents are chosen for the target and optimizationperformed. After an optimal transfection agent concentration is chosen,a RNA time-course of inhibition is performed with the lead siNAmolecule. In addition, a cell-plating format can be used to determineRNA inhibition.

Delivery of siNA to Cells

Cells (e.g., Jurkat, HeLa, or 293T cells) are seeded, for example, at1×10⁵ cells per well of a six-well dish in EGM-2 (BioWhittaker) the daybefore transfection. siNA (final concentration, for example 20 nM) andcationic lipid (e.g., final concentration 2 μg/ml) are complexed in EGMbasal media (Bio Whittaker) at 37° C. for 30 minutes in polystyrenetubes. Following vortexing, the complexed siNA is added to each well andincubated for the times indicated. For initial optimization experiments,cells are seeded, for example, at 1×10³ in 96 well plates and siNAcomplex added as described. Efficiency of delivery of siNA to cells isdetermined using a fluorescent siNA complexed with lipid. Cells in6-well dishes are incubated with siNA for 24 hours, rinsed with PBS andfixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptakeof siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, anddTTP, 10U RNase Inhibitor (Promega), 1.25U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/r×n) and normalizing to β-actin or GAPDH mRNA inparallel TAQMAN® reactions (real-time PCR monitoring of amplification).For each gene of interest an upper and lower primer and a fluorescentlylabeled probe are designed. Real time incorporation of SYBR Green I dyeinto a specific PCR product can be measured in glass capillary tubesusing a lightcyler. A standard curve is generated for each primer pairusing control cRNA. Values are represented as relative expression toGAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Animal Models Useful to Evaluate the Down-Regulation ofInterleukin and/or Interleukin Receptor Gene Expression

Evaluating the efficacy of anti-interleukin agents in animal models isan important prerequisite to human clinical trials. Allogeneic rejectionis the most common cause of corneal graft failure. King et al., 2000,Transplantation, 70, 1225-1233, describe a study investigating thekinetics of cytokine and chemokine mRNA expression before and after theonset of corneal graft rejection. Intracorneal cytokine and chemokinemRNA levels were investigated in the Brown Norway-Lewis inbred ratmodel, in which rejection onset is observed at 8/9 days after graftingin all animals. Nongrafted corneas and syngeneic (Lewis-Lewis) cornealtransplants were used as controls. Donor and recipient cornea wereexamined by quantitative competitive reverse transcription-polymerasechain reaction (RT-PCR) for hypoxanthine phosphoribosyltransferase(HPRT), CD3, CD25, interleukin (IL)-1beta, IL-1RA, IL-2, IL-6, IL-10,interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), transforminggrowth factor (TGF)-beta1, and macrophage inflammatory protein (MIP)-2and by RT-PCR for IL-4, IL-5, IL-12 p40, IL-13, TGF-beta.2, monocytechemotactic protein-1 (MCP-1), MIP-1alpha, MIP-1beta, and RANTES. Abiphasic expression of cytokine and chemokine mRNA was found aftertransplantation. During the early phase (days 3-9), there was anelevation of the majority of the cytokines examined, including IL-1beta,IL-6, IL-10, IL-12 p40, and MIP-2. There was no difference in cytokineexpression patterns between allogeneic or syngeneic recipients at thistime. In syngeneic recipients, cytokine levels reduced to pretransplantlevels by day 13, whereas levels of all cytokines rose after therejection onset in the allografts, including TGF-beta.1, TGF-beta.2, andIL-1RA. The T cell-derived cytokines IL-4, IL-13, and IFN-gamma weredetected only during the rejection phase in allogeneic recipients. Thus,there appears to be an early cytokine and chemokine response to thetransplantation process, evident in syngeneic and allogeneic grafts,that drives angiogenesis, leukocyte recruitment, and affects otherleukocyte functions. After an immune response has been generated,allogeneic rejection results in the expression of Th1 cytokines, Th2cytokines, and anti-inflammatory/Th3 cytokines. This animal model can beused to evaluate the efficacy of nucleic acid molecules of the inventiontargeting interleukin expression (e.g., phenotypic change, interleukinexpression etc.) toward therapeutic use in treating transplantrejection. Similarly, other animal models of transplant rejection as areknown in the art can be used to evaluate nucleic acid molecules (e.g.,siNA) of the invention toward therapeutic use.

Other animal models are useful in evaluating the role of interleukins inasthma. For example, Kuperman et al., 2002, Nature Medicine, 8, 885-9,describe an animal model of IL-13 mediated asthma response animal modelsof allergic asthma in which blockade of IL-13 markedly inhibitsallergen-induced asthma. Venkayya et al., 2002, Am J Respir Cell MolBiol., 26, 202-8 and Yang et al., 2001, Am J Respir Cell Mol Biol., 25,522-30 describe animal models of airway inflammation and airwayhyperresponsiveness (AHR) in which IL-4/IL-4R and IL-13 mediate asthma.These models can be used to evaluate the efficacy of siNA molecules ofthe invention targeting, for example, IL-4, IL-4R, IL-13, and/or IL-13Rfor use is treating asthma.

Example 9 RNAi Mediated Inhibition of Interleukin and/or InterleukinReceptor Expression in Cell Culture

Inhibition of Interleukin and/or Interleukin Receptor RNA ExpressionUsing siNA Targeting Interleukin and/or Interleukin Receptor RNA

siNA constructs (Table III) are tested for efficacy in reducinginterleukin and/or interleukin receptor RNA expression in, for example,Jurkat, HeLa, or 293T cells. Cells are plated approximately 24 hoursbefore transfection in 96-well plates at 5,000-7,500 cells/well, 100μl/well, such that at the time of transfection cells are 70-90%confluent. For transfection, annealed siNAs are mixed with thetransfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 0.5μl/well and incubated for 20 min. at room temperature. The siNAtransfection mixtures are added to cells to give a final siNAconcentration of 25 nM in a volume of 150 μl. Each siNA transfectionmixture is added to 3 wells for triplicate siNA treatments. Cells areincubated at 37° for 24 h in the continued presence of the siNAtransfection mixture. At 24 h, RNA is prepared from each well of treatedcells. The supernatants with the transfection mixtures are first removedand discarded, then the cells are lysed and RNA prepared from each well.Target gene expression following treatment is evaluated by RT-PCR forthe target gene and for a control gene (36B4, an RNA polymerase subunit)for normalization. The triplicate data is averaged and the standarddeviations determined for each treatment. Normalized data are graphedand the percent reduction of target mRNA by active siNAs in comparisonto their respective inverted control siNAs is determined.

In a non-limiting example, chemically modified siNA constructs (TableIII) were tested for efficacy as described above in reducing IL-4R RNAexpression in HeLa cells. Active siNAs were evaluated compared to amatched chemistry inverted control (IC), and a transfection control.Results are summarized in FIG. 22. FIG. 22 shows results for Stab 9/22(Table IV) siNA constructs targeting various sites in IL-4R mRNA. Asshown in FIG. 22, the active siNA constructs provide significantinhibition of IL-4R gene expression in cell culture experiments asdetermined by levels of IL-4R mRNA when compared to appropriatecontrols.

Example 10 Indications

The siNA molecule of the invention can be used to prevent, inhibit ortreat cancers and other proliferative conditions, viral infection,inflammatory disease, autoimmunity, respiratory disease, pulmonarydisease, cardiovascular disease, neurological disease, renal disease,ocular disease, liver disease, mitochondrial disease, endocrine disease,prion disease, reproduction related diseases and conditions, and/or anyother trait, disease or condition that is related to or will respond tothe levels of interleukin and/or interleukin receptor in a cell ortissue, alone or in combination with other therapies. Non-limitingexamples of respiratory diseases that can be treated using siNAmolecules of the invention (e.g., siNA molecules targeting IL-4, IL-4R,IL-13, and/or IL-13R include asthma, chronic obstructive pulmonarydisease or “COPD”, allergic rhinitis, sinusitis, pulmonaryvasoconstriction, inflammation, allergies, impeded respiration,respiratory distress syndrome, cystic fibrosis, pulmonary hypertension,pulmonary vasoconstriction, emphysema.

The use of anticholinergic agents, anti-inflammatories, bronchodilators,adenosine inhibitors, adenosine A1 receptor inhibitors, non-selective M3receptor antagonists such as atropine, ipratropium bromide and selectiveM3 receptor antagonists such as darifenacin and revatropate are allnon-limiting examples of agents that can be combined with or used inconjunction with the nucleic acid molecules (e.g. siNA molecules) of theinstant invention. Immunomodulators, chemotherapeutics,anti-inflammatory compounds, and anti-viral compounds are additionalnon-limiting examples of pharmaceutical agents that can be combined withor used in conjunction with the nucleic acid molecules (e.g. siNAmolecules) of the instant invention. Those skilled in the art willrecognize that other drugs, compounds and therapies can similarly bereadily combined with the nucleic acid molecules of the instantinvention (e.g. siRNA molecules) are hence within the scope of theinstant invention.

Example 11 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with an siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I interleukin and/or interleukin receptor Accession NumbersInterleukin Family NM_000575 Homo sapiens interleukin 1, alpha (IL1A),mRNA NM_000576 Homo sapiens interleukin 1, beta (IL1B), mRNA NM_012275Homo sapiens interleukin 1 family, member 5 (delta) (IL1F5), mRNANM_014440 Homo sapiens interleukin 1 family, member 6 (epsilon) (IL1F6),mRNA NM_014439 Homo sapiens interleukin 1 family, member 7 (zeta)(IL1F7), mRNA NM_014438 Homo sapiens interleukin 1 family, member 8(eta) (IL1F8), mRNA NM_019618 Homo sapiens interleukin 1 family, member9 (IL1F9), mRNA NM_032556 Homo sapiens interleukin 1 family, member 10(theta) (IL1F10), mRNA NM_000586 Homo sapiens interleukin 2 (IL2), mRNANM_000588 Homo sapiens interleukin 3 (colony-stimulating factor,multiple) (IL3), mRNA NM_000589 Homo sapiens interleukin 4 (IL4), mRNANM_000879 Homo sapiens interleukin 5 (colony-stimulating factor,eosinophil) (IL5), mRNA NM_000600 Homo sapiens interleukin 6(interferon, beta 2) (IL6), mRNA NM_000880 Homo sapiens interleukin 7(IL7), mRNA NM_000584 Homo sapiens interleukin 8 (IL8), mRNA NM_000590Homo sapiens interleukin 9 (IL9), mRNA NM_000572 Homo sapiensinterleukin 10 (IL10), mRNA NM_000641 Homo sapiens interleukin 11(IL11), mRNA NM_000882 Homo sapiens interleukin 12A (natural killer cellstimulatory factor 1, cytotoxic lymphocyte maturation factor 1, p35)(IL12A), mRNA NM_002187 Homo sapiens interleukin 12B (natural killercell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2,p40) (IL12B), mRNA NM_002188 Homo sapiens interleukin 13 (IL13), mRNAL15344 Homo sapiens interleukin 14 (IL14), mRNA NM_000585 Homo sapiensinterleukin 15 (IL15), mRNA NM_004513 Homo sapiens interleukin 16(lymphocyte chemoattractant factor) (IL16), mRNA NM_002190 Homo sapiensinterleukin 17 (cytotoxic T-lymphocyte- associated serine esterase 8)(IL17), mRNA NM_014443 Homo sapiens interleukin 17B (IL17B), mRNANM_013278 Homo sapiens interleukin 17C (IL17C), mRNA NM_138284 Homosapiens interleukin 17D (IL17D), mRNA NM_022789 Homo sapiens interleukin17E (IL17E), mRNA NM_052872 Homo sapiens interleukin 17F (IL17F), mRNANM_001562 Homo sapiens interleukin 18 (interferon-gamma-inducing factor)(IL18), mRNA NM_013371 Homo sapiens interleukin 19 (IL19), mRNANM_018724 Homo sapiens interleukin 20 (IL20), mRNA NM_021803 Homosapiens interleukin 21 (IL21 antisense), mRNA NM_020525 Homo sapiensinterleukin 22 (IL22), mRNA NM_016584 Homo sapiens interleukin 23, alphasubunit p19 (IL23A), mRNA NM_006850 Homo sapiens interleukin 24 (IL24),mRNA NM_018402 Homo sapiens interleukin 26 (IL26), mRNA AL365373 Homosapiens interleukin 27 (IL27), mRNA Interleukin Receptor FamilyNM_000877 Homo sapiens interleukin 1 receptor, type I (IL1R1), mRNANM_004633 Homo sapiens interleukin 1 receptor, type II (IL1R2), mRNANM_016232 Homo sapiens interleukin 1 receptor-like 1 (IL1RL1), mRNANM_003856 Homo sapiens interleukin 1 receptor-like 1 (IL1RL1), mRNANM_003854 Homo sapiens interleukin 1 receptor-like 2 (IL1RL2), mRNANM_000417 Homo sapiens interleukin 2 receptor, alpha (IL2RA), mRNANM_000878 Homo sapiens interleukin 2 receptor, beta (IL2RB), mRNANM_000206 Homo sapiens interleukin 2 receptor, gamma (severe combinedimmunodeficiency) (IL2RG), mRNA NM_002183 Homo sapiens interleukin 3receptor, alpha (low affinity) (IL3RA), mRNA NM_000418 Homo sapiensinterleukin 4 receptor (IL4R), mRNA NM_000564 Homo sapiens interleukin 5receptor, alpha (IL5RA), mRNA NM_000565 Homo sapiens interleukin 6receptor (IL6R), mRNA NM_002185 Homo sapiens interleukin 7 receptor(IL7R), mRNA NM_000634 Homo sapiens interleukin 8 receptor, alpha(IL8RA), mRNA NM_001557 Homo sapiens interleukin 8 receptor, beta(IL8RB), mRNA NM_002186 Homo sapiens interleukin 9 receptor (IL9R), mRNANM_001558 Homo sapiens interleukin 10 receptor, alpha (IL10RA), mRNANM_000628 Homo sapiens interleukin 10 receptor, beta (IL10RB), mRNANM_004512 Homo sapiens interleukin 11 receptor, alpha (IL11RA), mRNANM_005535 Homo sapiens interleukin 12 receptor, beta 1 (IL12RB1), mRNANM_001559 Homo sapiens interleukin 12 receptor, beta 2 (IL12RB2), mRNANM_001560 Homo sapiens interleukin 13 receptor, alpha 1 (IL13RA1), mRNANM_000640 Homo sapiens interleukin 13 receptor, alpha 2 (IL13RA2), mRNANM_002189 Homo sapiens interleukin 15 receptor, alpha (IL15RA), mRNANM_014339 Homo sapiens interleukin 17 receptor (IL17R), mRNA NM_032732Homo sapiens interleukin 17 receptor C (IL-17RC), mRNA NM_144640 Homosapiens interleukin 17 receptor E (IL-17RE), mRNA NM_018725 Homo sapiensinterleukin 17B receptor (IL17BR), mRNA NM_003855 Homo sapiensinterleukin 18 receptor 1 (IL18R1), mRNA NM_003853 Homo sapiensinterleukin 18 receptor accessory protein (IL18RAP), mRNA NM_014432 Homosapiens interleukin 20 receptor, alpha (IL20RA), mRNA NM_021798 Homosapiens interleukin 21 receptor (IL21 antisenseR), mRNA NM_021258 Homosapiens interleukin 22 receptor (IL22R), mRNA NM_144701 Homo sapiensinterleukin 23 receptor (IL23R), mRNA Interleukin Associated ProteinsNM_004514 Homo sapiens interleukin enhancer binding factor 1 (ILF1),mRNA NM_004515 Homo sapiens interleukin enhancer binding factor 2, 45 kD(ILF2), mRNA NM_012218 Homo sapiens interleukin enhancer binding factor3, 90 kD (ILF3), mRNA NM_004516 Homo sapiens interleukin enhancerbinding factor 3, 90 kD (ILF3), mRNA NM_016123 Homo sapiensinterleukin-1 receptor associated kinase 4 (IRAK4), mRNA NM_001569 Homosapiens interleukin-1 receptor-associated kinase 1 (IRAK1), mRNANM_001570 Homo sapiens interleukin-1 receptor-associated kinase 2(IRAK2), mRNA NM_007199 Homo sapiens interleukin-1 receptor-associatedkinase 3 (IRAK3), mRNA NM_134470 Homo sapiens interleukin 1 receptoraccessory protein (IL1RAP), mRNA NM_002182 Homo sapiens interleukin 1receptor accessory protein (IL1RAP), mRNA NM_014271 Homo sapiensinterleukin 1 receptor accessory protein-like 1 (IL1RAPL1), mRNANM_017416 Homo sapiens interleukin 1 receptor accessory protein-like 2(IL1RAPL2), mRNA NM_000577 Homo sapiens interleukin 1 receptorantagonist (IL1RN), mRNA NM_002184 Homo sapiens interleukin 6 signaltransducer (gp130, oncostatin M receptor) (IL6ST), mRNA NM_005699 Homosapiens interleukin 18 binding protein (IL18BP), mRNA

TABLE II Interleukin and Interleukin receptor siNA and Target SequencesSeq Seq Seq Pos Seq ID UPos Upper seq ID LPos Lower seq ID IL2RGNM_000206 3 AGAGCAAGCGCCAUGUUGA 1 3 AGAGCAAGCGCCAUGUUGA 1 25UCAACAUGGCGCUUGCUCU 82 21 AAGCCAUCAUUACCAUUCA 2 21 AAGCCAUCAUUACCAUUCA 243 UGAAUGGUAAUGAUGGCUU 83 39 ACAUCCCUCUUAUUCCUGC 3 39ACAUCCCUCUUAUUCCUGC 3 61 GCAGGAAUAAGAGGGAUGU 84 57 CAGCUGCCCCUGCUGGGAG 457 CAGCUGCCCCUGCUGGGAG 4 79 CUCCCAGCAGGGGCAGCUG 85 75GUGGGGCUGAACACGACAA 5 75 GUGGGGCUGAACACGACAA 5 97 UUGUCGUGUUCAGCCCCAC 8693 AUUCUGACGCCCAAUGGGA 6 93 AUUCUGACGCCCAAUGGGA 6 115UCCCAUUGGGCGUCAGAAU 87 111 AAUGAAGACACCACAGCUG 7 111 AAUGAAGACACCACAGCUG7 133 CAGCUGUGGUGUCUUCAUU 88 129 GAUUUCUUCCUGACCACUA 8 129GAUUUCUUCCUGACCACUA 8 151 UAGUGGUCAGGAAGAAAUC 89 147 AUGCCCACUGACUCCCUCA9 147 AUGCCCACUGACUCCCUCA 9 169 UGAGGGAGUCAGUGGGCAU 90 165AGUGUUUCCACUCUGCCCC 10 165 AGUGUUUCCACUCUGCCCC 10 187GGGGCAGAGUGGAAACACU 91 183 CUCCCAGAGGUUCAGUGUU 11 183CUCCCAGAGGUUCAGUGUU 11 205 AACACUGAACCUCUGGGAG 92 201UUUGUGUUCAAUGUCGAGU 12 201 UUUGUGUUCAAUGUCGAGU 12 223ACUCGACAUUGAACACAAA 93 219 UACAUGAAUUGCACUUGGA 13 219UACAUGAAUUGCACUUGGA 13 241 UCCAAGUGCAAUUCAUGUA 94 237AACAGCAGCUCUGAGCCCC 14 237 AACAGCAGCUCUGAGCCCC 14 259GGGGCUCAGAGCUGCUGUU 95 255 CAGCCUACCAACCUCACUC 15 255CAGCCUACCAACCUCACUC 15 277 GAGUGAGGUUGGUAGGCUG 96 273CUGCAUUAUUGGUACAAGA 16 273 CUGCAUUAUUGGUACAAGA 16 295UCUUGUACCAAUAAUGCAG 97 291 AACUCGGAUAAUGAUAAAG 17 291AACUCGGAUAAUGAUAAAG 17 313 CUUUAUCAUUAUCCGAGUU 98 309GUCCAGAAGUGCAGCCACU 18 309 GUCCAGAAGUGCAGCCACU 18 331AGUGGCUGCACUUCUGGAC 99 327 UAUCUAUUCUCUGAAGAAA 19 327UAUCUAUUCUCUGAAGAAA 19 349 UUUCUUCAGAGAAUAGAUA 100 345AUCACUUCUGGCUGUCAGU 20 345 AUCACUUCUGGCUGUCAGU 20 367ACUGACAGCCAGAAGUGAU 101 363 UUGCAAAAAAAGGAGAUCC 21 363UUGCAAAAAAAGGAGAUCC 21 385 GGAUCUCCUUUUUUUGCAA 102 381CACCUCUACCAAACAUUUG 22 381 CACCUCUACCAAACAUUUG 22 403CAAAUGUUUGGUAGAGGUG 103 399 GUUGUUCAGCUCCAGGACC 23 399GUUGUUCAGCUCCAGGACC 23 421 GGUCCUGGAGCUGAACAAC 104 417CCACGGGAACCCAGGAGAC 24 417 CCACGGGAACCCAGGAGAC 24 439GUCUCCUGGGUUCCCGUGG 105 435 CAGGCCACACAGAUGCUAA 25 435CAGGCCACACAGAUGCUAA 25 457 UUAGCAUCUGUGUGGCCUG 106 453AAACUGCAGAAUCUGGUGA 26 453 AAACUGCAGAAUCUGGUGA 26 475UCACCAGAUUCUGCAGUUU 107 471 AUCCCCUGGGCUCCAGAGA 27 471AUCCCCUGGGCUCCAGAGA 27 493 UCUCUGGAGCCCAGGGGAU 108 489AACCUAACACUUCACAAAC 28 489 AACCUAACACUUCACAAAC 28 511GUUUGUGAAGUGUUAGGUU 109 507 CUGAGUGAAUCCCAGCUAG 29 507CUGAGUGAAUCCCAGCUAG 29 529 CUAGCUGGGAUUCACUCAG 110 525GAACUGAACUGGAACAACA 30 525 GAACUGAACUGGAACAACA 30 547UGUUGUUCCAGUUCAGUUC 111 543 AGAUUCUUGAACCACUGUU 31 543AGAUUCUUGAACCACUGUU 31 565 AACAGUGGUUCAAGAAUCU 112 561UUGGAGCACUUGGUGCAGU 32 561 UUGGAGCACUUGGUGCAGU 32 583ACUGCACCAAGUGCUCCAA 113 579 UACCGGACUGACUGGGACC 33 579UACCGGACUGACUGGGACC 33 601 GGUCCCAGUCAGUCCGGUA 114 597CACAGCUGGACUGAACAAU 34 597 CACAGCUGGACUGAACAAU 34 619AUUGUUCAGUCCAGCUGUG 115 615 UCAGUGGAUUAUAGACAUA 35 615UCAGUGGAUUAUAGACAUA 35 637 UAUGUCUAUAAUCCACUGA 116 633AAGUUCUCCUUGCCUAGUG 36 633 AAGUUCUCCUUGCCUAGUG 36 655CACUAGGCAAGGAGAACUU 117 651 GUGGAUGGGCAGAAACGCU 37 651GUGGAUGGGCAGAAACGCU 37 673 AGCGUUUCUGCCCAUCCAC 118 669UACACGUUUCGUGUUCGGA 38 669 UACACGUUUCGUGUUCGGA 38 691UCCGAACACGAAACGUGUA 119 687 AGCCGCUUUAACCCACUCU 39 687AGCCGCUUUAACCCACUCU 39 709 AGAGUGGGUUAAAGCGGCU 120 705UGUGGAAGUGCUCAGCAUU 40 705 UGUGGAAGUGCUCAGCAUU 40 727AAUGCUGAGCACUUCCACA 121 723 UGGAGUGAAUGGAGCCACC 41 723UGGAGUGAAUGGAGCCACC 41 745 GGUGGCUCCAUUCACUCCA 122 741CCAAUCCACUGGGGGAGCA 42 741 CCAAUCCACUGGGGGAGCA 42 763UGCUCCCCCAGUGGAUUGG 123 759 AAUACUUCAAAAGAGAAUC 43 759AAUACUUCAAAAGAGAAUC 43 781 GAUUCUCUUUUGAAGUAUU 124 777CCUUUCCUGUUUGCAUUGG 44 777 CCUUUCCUGUUUGCAUUGG 44 799CCAAUGCAAACAGGAAAGG 125 795 GAAGCCGUGGUUAUCUCUG 45 795GAAGCCGUGGUUAUCUCUG 45 817 CAGAGAUAACCACGGCUUC 126 813GUUGGCUCCAUGGGAUUGA 46 813 GUUGGCUCCAUGGGAUUGA 46 835UCAAUCCCAUGGAGCCAAC 127 831 AUUAUCAGCCUUCUCUGUG 47 831AUUAUCAGCCUUCUCUGUG 47 853 CACAGAGAAGGCUGAUAAU 128 849GUGUAUUUCUGGCUGGAAC 48 849 GUGUAUUUCUGGCUGGAAC 48 871GUUCCAGCCAGAAAUACAC 129 867 CGGACGAUGCCCCGAAUUC 49 867CGGACGAUGCCCCGAAUUC 49 889 GAAUUCGGGGCAUCGUCCG 130 885CCCACCCUGAAGAACCUAG 50 885 CCCACCCUGAAGAACCUAG 50 907CUAGGUUCUUCAGGGUGGG 131 903 GAGGAUCUUGUUACUGAAU 51 903GAGGAUCUUGUUACUGAAU 51 925 AUUCAGUAACAAGAUCCUC 132 921UACCACGGGAACUUUUCGG 52 921 UACCACGGGAACUUUUCGG 52 943CCGAAAAGUUCCCGUGGUA 133 939 GCCUGGAGUGGUGUGUCUA 53 939GCCUGGAGUGGUGUGUCUA 53 961 UAGACACACCACUCCAGGC 134 957AAGGGACUGGCUGAGAGUC 54 957 AAGGGACUGGCUGAGAGUC 54 979GACUCUCAGCCAGUCCCUU 135 975 CUGCAGCCAGACUACAGUG 55 975CUGCAGCCAGACUACAGUG 55 997 CACUGUAGUCUGGCUGCAG 136 993GAACGACUCUGCCUCGUCA 56 993 GAACGACUCUGCCUCGUCA 56 1015UGACGAGGCAGAGUCGUUC 137 1011 AGUGAGAUUCCCCCAAAAG 57 1011AGUGAGAUUCCCCCAAAAG 57 1033 CUUUUGGGGGAAUCUCACU 138 1029GGAGGGGCCCUUGGGGAGG 58 1029 GGAGGGGCCCUUGGGGAGG 58 1051CCUCCCCAAGGGCCCCUCC 139 1047 GGGCCUGGGGCCUCCCCAU 59 1047GGGCCUGGGGCCUCCCCAU 59 1069 AUGGGGAGGCCCCAGGCCC 140 1065UGCAACCAGCAUAGCCCCU 60 1065 UGCAACCAGCAUAGCCCCU 60 1087AGGGGCUAUGCUGGUUGCA 141 1083 UACUGGGCCCCCCCAUGUU 61 1083UACUGGGCCCCCCCAUGUU 61 1105 AACAUGGGGGGGCCCAGUA 142 1101UACACCCUAAAGCCUGAAA 62 1101 UACACCCUAAAGCCUGAAA 62 1123UUUCAGGCUUUAGGGUGUA 143 1119 ACCUGAACCCCAAUCCUCU 63 1119ACCUGAACCCCAAUCCUCU 63 1141 AGAGGAUUGGGGUUCAGGU 144 1137UGACAGAAGAACCCCAGGG 64 1137 UGACAGAAGAACCCCAGGG 64 1159CCCUGGGGUUCUUCUGUCA 145 1155 GUCCUGUAGCCCUAAGUGG 65 1155GUCCUGUAGCCCUAAGUGG 65 1177 CCACUUAGGGCUACAGGAC 146 1173GUACUAACUUUCCUUCAUU 66 1173 GUACUAACUUUCCUUCAUU 66 1195AAUGAAGGAAAGUUAGUAC 147 1191 UCAACCCACCUGCGUCUCA 67 1191UCAACCCACCUGCGUCUCA 67 1213 UGAGACGCAGGUGGGUUGA 148 1209AUACUCACCUCACCCCACU 68 1209 AUACUCACCUCACCCCACU 68 1231AGUGGGGUGAGGUGAGUAU 149 1227 UGUGGCUGAUUUGGAAUUU 69 1227UGUGGCUGAUUUGGAAUUU 69 1249 AAAUUCCAAAUCAGCCACA 150 1245UUGUGCCCCCAUGUAAGCA 70 1245 UUGUGCCCCCAUGUAAGCA 70 1267UGCUUACAUGGGGGCACAA 151 1263 ACCCCUUCAUUUGGCAUUC 71 1263ACCCCUUCAUUUGGCAUUC 71 1285 GAAUGCCAAAUGAAGGGGU 152 1281CCCCACUUGAGAAUUACCC 72 1281 CCCCACUUGAGAAUUACCC 72 1303GGGUAAUUCUCAAGUGGGG 153 1299 CUUUUGCCCCGAACAUGUU 73 1299CUUUUGCCCCGAACAUGUU 73 1321 AACAUGUUCGGGGCAAAAG 154 1317UUUUCUUCUCCCUCAGUCU 74 1317 UUUUCUUCUCCCUCAGUCU 74 1339AGACUGAGGGAGAAGAAAA 155 1335 UGGCCCUUCCUUUUCGCAG 75 1335UGGCCCUUCCUUUUCGCAG 75 1357 CUGCGAAAAGGAAGGGCCA 156 1353GGAUUCUUCCUCCCUCCCU 76 1353 GGAUUCUUCCUCCCUCCCU 76 1375AGGGAGGGAGGAAGAAUCC 157 1371 UCUUUCCCUCCCUUCCUCU 77 1371UCUUUCCCUCCCUUCCUCU 77 1393 AGAGGAAGGGAGGGAAAGA 158 1389UUUCCAUCUACCCUCCGAU 78 1389 UUUCCAUCUACCCUCCGAU 78 1411AUCGGAGGGUAGAUGGAAA 159 1407 UUGUUCCUGAACCGAUGAG 79 1407UUGUUCCUGAACCGAUGAG 79 1429 CUCAUCGGUUCAGGAACAA 160 1425GAAAUAAAGUUUCUGUUGA 80 1425 GAAAUAAAGUUUCUGUUGA 80 1447UCAACAGAAACUUUAUUUC 161 1431 AAGUUUCUGUUGAUAAUCA 81 1431AAGUUUCUGUUGAUAAUCA 81 1453 UGAUUAUCAACAGAAACUU 162 IL4 NM_000589 3CUAUGCAAAGCAAAAAGCC 163 3 CUAUGCAAAGCAAAAAGCC 163 25 GGCUUUUUGCUUUGCAUAG214 21 CAGCAGCAGCCCCAAGCUG 164 21 CAGCAGCAGCCCCAAGCUG 164 43CAGCUUGGGGCUGCUGCUG 215 39 GAUAAGAUUAAUCUAAAGA 165 39GAUAAGAUUAAUCUAAAGA 165 61 UCUUUAGAUUAAUCUUAUC 216 57AGCAAAUUAUGGUGUAAUU 166 57 AGCAAAUUAUGGUGUAAUU 166 79AAUUACACCAUAAUUUGCU 217 75 UUCCUAUGCUGAAACUUUG 167 75UUCCUAUGCUGAAACUUUG 167 97 CAAAGUUUCAGCAUAGGAA 218 93GUAGUUAAUUUUUUAAAAA 168 93 GUAGUUAAUUUUUUAAAAA 168 115UUUUUAAAAAAUUAACUAC 219 111 AGGUUUCAUUUUCCUAUUG 169 111AGGUUUCAUUUUCCUAUUG 169 133 CAAUAGGAAAAUGAAACCU 220 129GGUCUGAUUUCACAGGAAC 170 129 GGUCUGAUUUCACAGGAAC 170 151GUUCCUGUGAAAUCAGACC 221 147 CAUUUUACCUGUUUGUGAG 171 147CAUUUUACCUGUUUGUGAG 171 169 CUCACAAACAGGUAAAAUG 222 165GGCAUUUUUUCUCCUGGAA 172 165 GGCAUUUUUUCUCCUGGAA 172 187UUCCAGGAGAAAAAAUGCC 223 183 AGAGAGGUGCUGAUUGGCC 173 183AGAGAGGUGCUGAUUGGCC 173 205 GGCCAAUCAGCACCUCUCU 224 201CCCAAGUGACUGACAAUCU 174 201 CCCAAGUGACUGACAAUCU 174 223AGAUUGUCAGUCACUUGGG 225 219 UGGUGUAACGAAAAUUUCC 175 219UGGUGUAACGAAAAUUUCC 175 241 GGAAAUUUUCGUUACACCA 226 237CAAUGUAAACUCAUUUUCC 176 237 CAAUGUAAACUCAUUUUCC 176 259GGAAAAUGAGUUUACAUUG 227 255 CCUCGGUUUCAGCAAUUUU 177 255CCUCGGUUUCAGCAAUUUU 177 277 AAAAUUGCUGAAACCGAGG 228 273UAAAUCUAUAUAUAGAGAU 178 273 UAAAUCUAUAUAUAGAGAU 178 295AUCUCUAUAUAUAGAUUUA 229 291 UAUCUUUGUCAGCAUUGCA 179 291UAUCUUUGUCAGCAUUGCA 179 313 UGCAAUGCUGACAAAGAUA 230 309AUCGUUAGCUUCUCCUGAU 180 309 AUCGUUAGCUUCUCCUGAU 180 331AUCAGGAGAAGCUAACGAU 231 327 UAAACUAAUUGCCUCACAU 181 327UAAACUAAUUGCCUCACAU 181 349 AUGUGAGGCAAUUAGUUUA 232 345UUGUCACUGCAAAUCGACA 182 345 UUGUCACUGCAAAUCGACA 182 367UGUCGAUUUGCAGUGACAA 233 363 ACCUAUUAAUGGGUCUCAC 183 363ACCUAUUAAUGGGUCUCAC 183 385 GUGAGACCCAUUAAUAGGU 234 381CCUCCCAACUGCUUCCCCC 184 381 CCUCCCAACUGCUUCCCCC 184 403GGGGGAAGCAGUUGGGAGG 235 399 CUCUGUUCUUCCUGCUAGC 185 399CUCUGUUCUUCCUGCUAGC 185 421 GCUAGCAGGAAGAACAGAG 236 417CAUGUGCCGGCAACUUUGU 186 417 CAUGUGCCGGCAACUUUGU 186 439ACAAAGUUGCCGGCACAUG 237 435 UCCACGGACACAAGUGCGA 187 435UCCACGGACACAAGUGCGA 187 457 UCGCACUUGUGUCCGUGGA 238 453AUAUCACCUUACAGGAGAU 188 453 AUAUCACCUUACAGGAGAU 188 475AUCUCCUGUAAGGUGAUAU 239 471 UCAUCAAAACUUUGAACAG 189 471UCAUCAAAACUUUGAACAG 189 493 CUGUUCAAAGUUUUGAUGA 240 489GCCUCACAGAGCAGAAGAC 190 489 GCCUCACAGAGCAGAAGAC 190 511GUCUUCUGCUCUGUGAGGC 241 507 CUCUGUGCACCGAGUUGAC 191 507CUCUGUGCACCGAGUUGAC 191 529 GUCAACUCGGUGCACAGAG 242 525CCGUAACAGACAUCUUUGC 192 525 CCGUAACAGACAUCUUUGC 192 547GCAAAGAUGUCUGUUACGG 243 543 CUGCCUCCAAGAACACAAC 193 543CUGCCUCCAAGAACACAAC 193 565 GUUGUGUUCUUGGAGGCAG 244 561CUGAGAAGGAAACCUUCUG 194 561 CUGAGAAGGAAACCUUCUG 194 583CAGAAGGUUUCCUUCUCAG 245 579 GCAGGGCUGCGACUGUGCU 195 579GCAGGGCUGCGACUGUGCU 195 601 AGCACAGUCGCAGCCCUGC 246 597UCCGGCAGUUCUACAGCCA 196 597 UCCGGCAGUUCUACAGCCA 196 619UGGCUGUAGAACUGCCGGA 247 615 ACCAUGAGAAGGACACUCG 197 615ACCAUGAGAAGGACACUCG 197 637 CGAGUGUCCUUCUCAUGGU 248 633GCUGCCUGGGUGCGACUGC 198 633 GCUGCCUGGGUGCGACUGC 198 655GCAGUCGCACCCAGGCAGC 249 651 CACAGCAGUUCCACAGGCA 199 651CACAGCAGUUCCACAGGCA 199 673 UGCCUGUGGAACUGCUGUG 250 669ACAAGCAGCUGAUCCGAUU 200 669 ACAAGCAGCUGAUCCGAUU 200 691AAUCGGAUCAGCUGCUUGU 251 687 UCCUGAAACGGCUCGACAG 201 687UCCUGAAACGGCUCGACAG 201 709 CUGUCGAGCCGUUUCAGGA 252 705GGAACCUCUGGGGCCUGGC 202 705 GGAACCUCUGGGGCCUGGC 202 727GCCAGGCCCCAGAGGUUCC 253 723 CGGGCUUGAAUUCCUGUCC 203 723CGGGCUUGAAUUCCUGUCC 203 745 GGACAGGAAUUCAAGCCCG 254 741CUGUGAAGGAAGCCAACCA 204 741 CUGUGAAGGAAGCCAACCA 204 763UGGUUGGCUUCCUUCACAG 255 759 AGAGUACGUUGGAAAACUU 205 759AGAGUACGUUGGAAAACUU 205 781 AAGUUUUCCAACGUACUCU 256 777UCUUGGAAAGGCUAAAGAC 206 777 UCUUGGAAAGGCUAAAGAC 206 799GUCUUUAGCCUUUCCAAGA 257 795 CGAUCAUGAGAGAGAAAUA 207 795CGAUCAUGAGAGAGAAAUA 207 817 UAUUUCUCUCUCAUGAUCG 258 813AUUCAAAGUGUUCGAGCUG 208 813 AUUCAAAGUGUUCGAGCUG 208 835CAGCUCGAACACUUUGAAU 259 831 GAAUAUUUUAAUUUAUGAG 209 831GAAUAUUUUAAUUUAUGAG 209 853 CUCAUAAAUUAAAAUAUUC 260 849GUUUUUGAUAGCUUUAUUU 210 849 GUUUUUGAUAGCUUUAUUU 210 871AAAUAAAGCUAUCAAAAAC 261 867 UUUUAAGUAUUUAUAUAUU 211 867UUUUAAGUAUUUAUAUAUU 211 889 AAUAUAUAAAUACUUAAAA 262 885UUAUAACUCAUCAUAAAAU 212 885 UUAUAACUCAUCAUAAAAU 212 907AUUUUAUGAUGAGUUAUAA 263 901 AAUAAAGUAUAUAUAGAAU 213 901AAUAAAGUAUAUAUAGAAU 213 923 AUUCUAUAUAUACUUUAUU 264 IL4R NM_000418 3CGAAUGGAGCAGGGGCGCG 265 3 CGAAUGGAGCAGGGGCGCG 265 25 CGCGCCCCUGCUCCAUUCG465 21 GCAGAUAAUUAAAGAUUUA 266 21 GCAGAUAAUUAAAGAUUUA 266 43UAAAUCUUUAAUUAUCUGC 466 39 ACACACAGCUGGAAGAAAU 267 39ACACACAGCUGGAAGAAAU 267 61 AUUUCUUCCAGCUGUGUGU 467 57UCAUAGAGAAGCCGGGCGU 268 57 UCAUAGAGAAGCCGGGCGU 268 79ACGCCCGGCUUCUCUAUGA 468 75 UGGUGGCUCAUGCCUAUAA 269 75UGGUGGCUCAUGCCUAUAA 269 97 UUAUAGGCAUGAGCCACCA 469 93AUCCCAGCACUUUUGGAGG 270 93 AUCCCAGCACUUUUGGAGG 270 115CCUCCAAAAGUGCUGGGAU 470 111 GCUGAGGCGGGCAGAUCAC 271 111GCUGAGGCGGGCAGAUCAC 271 133 GUGAUCUGCCCGCCUCAGC 471 129CUUGAGAUCAGGAGUUCGA 272 129 CUUGAGAUCAGGAGUUCGA 272 151UCGAACUCCUGAUCUCAAG 472 147 AGACCAGCCUGGUGCCUUG 273 147AGACCAGCCUGGUGCCUUG 273 169 CAAGGCACCAGGCUGGUCU 473 165GGCAUCUCCCAAUGGGGUG 274 165 GGCAUCUCCCAAUGGGGUG 274 187CACCCCAUUGGGAGAUGCC 474 183 GGCUUUGCUCUGGGCUCCU 275 183GGCUUUGCUCUGGGCUCCU 275 205 AGGAGCCCAGAGCAAAGCC 475 201UGUUCCCUGUGAGCUGCCU 276 201 UGUUCCCUGUGAGCUGCCU 276 223AGGCAGCUCACAGGGAACA 476 219 UGGUCCUGCUGCAGGUGGC 277 219UGGUCCUGCUGCAGGUGGC 277 241 GCCACCUGCAGCAGGACCA 477 237CAAGCUCUGGGAACAUGAA 278 237 CAAGCUCUGGGAACAUGAA 278 259UUCAUGUUCCCAGAGCUUG 478 255 AGGUCUUGCAGGAGCCCAC 279 255AGGUCUUGCAGGAGCCCAC 279 277 GUGGGCUCCUGCAAGACCU 479 273CCUGCGUCUCCGACUACAU 280 273 CCUGCGUCUCCGACUACAU 280 295AUGUAGUCGGAGACGCAGG 480 291 UGAGCAUCUCUACUUGCGA 281 291UGAGCAUCUCUACUUGCGA 281 313 UCGCAAGUAGAGAUGCUCA 481 309AGUGGAAGAUGAAUGGUCC 282 309 AGUGGAAGAUGAAUGGUCC 282 331GGACCAUUCAUCUUCCACU 482 327 CCACCAAUUGCAGCACCGA 283 327CCACCAAUUGCAGCACCGA 283 349 UCGGUGCUGCAAUUGGUGG 483 345AGCUCCGCCUGUUGUACCA 284 345 AGCUCCGCCUGUUGUACCA 284 367UGGUACAACAGGCGGAGCU 484 363 AGCUGGUUUUUCUGCUCUC 285 363AGCUGGUUUUUCUGCUCUC 285 385 GAGAGCAGAAAAACCAGCU 485 381CCGAAGCCCACACGUGUAU 286 381 CCGAAGCCCACACGUGUAU 286 403AUACACGUGUGGGCUUCGG 486 399 UCCCUGAGAACAACGGAGG 287 399UCCCUGAGAACAACGGAGG 287 421 CCUCCGUUGUUCUCAGGGA 487 417GCGCGGGGUGCGUGUGCCA 288 417 GCGCGGGGUGCGUGUGCCA 288 439UGGCACACGCACCCCGCGC 488 435 ACCUGCUCAUGGAUGACGU 289 435ACCUGCUCAUGGAUGACGU 289 457 ACGUCAUCCAUGAGCAGGU 489 453UGGUCAGUGCGGAUAACUA 290 453 UGGUCAGUGCGGAUAACUA 290 475UAGUUAUCCGCACUGACCA 490 471 AUACACUGGACCUGUGGGC 291 471AUACACUGGACCUGUGGGC 291 493 GCCCACAGGUCCAGUGUAU 491 489CUGGGCAGCAGCUGCUGUG 292 489 CUGGGCAGCAGCUGCUGUG 292 511CACAGCAGCUGCUGCCCAG 492 507 GGAAGGGCUCCUUCAAGCC 293 507GGAAGGGCUCCUUCAAGCC 293 529 GGCUUGAAGGAGCCCUUCC 493 525CCAGCGAGCAUGUGAAACC 294 525 CCAGCGAGCAUGUGAAACC 294 547GGUUUCACAUGCUCGCUGG 494 543 CCAGGGCCCCAGGAAACCU 295 543CCAGGGCCCCAGGAAACCU 295 565 AGGUUUCCUGGGGCCCUGG 495 561UGACAGUUCACACCAAUGU 296 561 UGACAGUUCACACCAAUGU 296 583ACAUUGGUGUGAACUGUCA 496 579 UCUCCGACACUCUGCUGCU 297 579UCUCCGACACUCUGCUGCU 297 601 AGCAGCAGAGUGUCGGAGA 497 597UGACCUGGAGCAACCCGUA 298 597 UGACCUGGAGCAACCCGUA 298 619UACGGGUUGCUCCAGGUCA 498 615 AUCCCCCUGACAAUUACCU 299 615AUCCCCCUGACAAUUACCU 299 637 AGGUAAUUGUCAGGGGGAU 499 633UGUAUAAUCAUCUCACCUA 300 633 UGUAUAAUCAUCUCACCUA 300 655UAGGUGAGAUGAUUAUACA 500 651 AUGCAGUCAACAUUUGGAG 301 651AUGCAGUCAACAUUUGGAG 301 673 CUCCAAAUGUUGACUGCAU 501 669GUGAAAACGACCCGGCAGA 302 669 GUGAAAACGACCCGGCAGA 302 691UCUGCCGGGUCGUUUUCAC 502 687 AUUUCAGAAUCUAUAACGU 303 687AUUUCAGAAUCUAUAACGU 303 709 ACGUUAUAGAUUCUGAAAU 503 705UGACCUACCUAGAACCCUC 304 705 UGACCUACCUAGAACCCUC 304 727GAGGGUUCUAGGUAGGUCA 504 723 CCCUCCGCAUCGCAGCCAG 305 723CCCUCCGCAUCGCAGCCAG 305 745 CUGGCUGCGAUGCGGAGGG 505 741GCACCCUGAAGUCUGGGAU 306 741 GCACCCUGAAGUCUGGGAU 306 763AUCCCAGACUUCAGGGUGC 506 759 UUUCCUACAGGGCACGGGU 307 759UUUCCUACAGGGCACGGGU 307 781 ACCCGUGCCCUGUAGGAAA 507 777UGAGGGCCUGGGCUCAGUG 308 777 UGAGGGCCUGGGCUCAGUG 308 799CACUGAGCCCAGGCCCUCA 508 795 GCUAUAACACCACCUGGAG 309 795GCUAUAACACCACCUGGAG 309 817 CUCCAGGUGGUGUUAUAGC 509 813GUGAGUGGAGCCCCAGCAC 310 813 GUGAGUGGAGCCCCAGCAC 310 835GUGCUGGGGCUCCACUCAC 510 831 CCAAGUGGCACAACUCCUA 311 831CCAAGUGGCACAACUCCUA 311 853 UAGGAGUUGUGCCACUUGG 511 849ACAGGGAGCCCUUCGAGCA 312 849 ACAGGGAGCCCUUCGAGCA 312 871UGCUCGAAGGGCUCCCUGU 512 867 AGCACCUCCUGCUGGGCGU 313 867AGCACCUCCUGCUGGGCGU 313 889 ACGCCCAGCAGGAGGUGCU 513 885UCAGCGUUUCCUGCAUUGU 314 885 UCAGCGUUUCCUGCAUUGU 314 907ACAAUGCAGGAAACGCUGA 514 903 UCAUCCUGGCCGUCUGCCU 315 903UCAUCCUGGCCGUCUGCCU 315 925 AGGCAGACGGCCAGGAUGA 515 921UGUUGUGCUAUGUCAGCAU 316 921 UGUUGUGCUAUGUCAGCAU 316 943AUGCUGACAUAGCACAACA 516 939 UCACCAAGAUUAAGAAAGA 317 939UCACCAAGAUUAAGAAAGA 317 961 UCUUUCUUAAUCUUGGUGA 517 957AAUGGUGGGAUCAGAUUCC 318 957 AAUGGUGGGAUCAGAUUCC 318 979GGAAUCUGAUCCCACCAUU 518 975 CCAACCCAGCCCGCAGCCG 319 975CCAACCCAGCCCGCAGCCG 319 997 CGGCUGCGGGCUGGGUUGG 519 993GCCUCGUGGCUAUAAUAAU 320 993 GCCUCGUGGCUAUAAUAAU 320 1015AUUAUUAUAGCCACGAGGC 520 1011 UCCAGGAUGCUCAGGGGUC 321 1011UCCAGGAUGCUCAGGGGUC 321 1033 GACCCCUGAGCAUCCUGGA 521 1029CACAGUGGGAGAAGCGGUC 322 1029 CACAGUGGGAGAAGCGGUC 322 1051GACCGCUUCUCCCACUGUG 522 1047 CCCGAGGCCAGGAACCAGC 323 1047CCCGAGGCCAGGAACCAGC 323 1069 GCUGGUUCCUGGCCUCGGG 523 1065CCAAGUGCCCACACUGGAA 324 1065 CCAAGUGCCCACACUGGAA 324 1087UUCCAGUGUGGGCACUUGG 524 1083 AGAAUUGUCUUACCAAGCU 325 1083AGAAUUGUCUUACCAAGCU 325 1105 AGCUUGGUAAGACAAUUCU 525 1101UCUUGCCCUGUUUUCUGGA 326 1101 UCUUGCCCUGUUUUCUGGA 326 1123UCCAGAAAACAGGGCAAGA 526 1119 AGCACAACAUGAAAAGGGA 327 1119AGCACAACAUGAAAAGGGA 327 1141 UCCCUUUUCAUGUUGUGCU 527 1137AUGAAGAUCCUCACAAGGC 328 1137 AUGAAGAUCCUCACAAGGC 328 1159GCCUUGUGAGGAUCUUCAU 528 1155 CUGCCAAAGAGAUGCCUUU 329 1155CUGCCAAAGAGAUGCCUUU 329 1177 AAAGGCAUCUCUUUGGCAG 529 1173UCCAGGGCUCUGGAAAAUC 330 1173 UCCAGGGCUCUGGAAAAUC 330 1195GAUUUUCCAGAGCCCUGGA 530 1191 CAGCAUGGUGCCCAGUGGA 331 1191CAGCAUGGUGCCCAGUGGA 331 1213 UCCACUGGGCACCAUGCUG 531 1209AGAUCAGCAAGACAGUCCU 332 1209 AGAUCAGCAAGACAGUCCU 332 1231AGGACUGUCUUGCUGAUCU 532 1227 UCUGGCCAGAGAGCAUCAG 333 1227UCUGGCCAGAGAGCAUCAG 333 1249 CUGAUGCUCUCUGGCCAGA 533 1245GCGUGGUGCGAUGUGUGGA 334 1245 GCGUGGUGCGAUGUGUGGA 334 1267UCCACACAUCGCACCACGC 534 1263 AGUUGUUUGAGGCCCCGGU 335 1263AGUUGUUUGAGGCCCCGGU 335 1285 ACCGGGGCCUCAAACAACU 535 1281UGGAGUGUGAGGAGGAGGA 336 1281 UGGAGUGUGAGGAGGAGGA 336 1303UCCUCCUCCUCACACUCCA 536 1299 AGGAGGUAGAGGAAGAAAA 337 1299AGGAGGUAGAGGAAGAAAA 337 1321 UUUUCUUCCUCUACCUCCU 537 1317AAGGGAGCUUCUGUGCAUC 338 1317 AAGGGAGCUUCUGUGCAUC 338 1339GAUGCACAGAAGCUCCCUU 538 1335 CGCCUGAGAGCAGCAGGGA 339 1335CGCCUGAGAGCAGCAGGGA 339 1357 UCCCUGCUGCUCUCAGGCG 539 1353AUGACUUCCAGGAGGGAAG 340 1353 AUGACUUCCAGGAGGGAAG 340 1375CUUCCCUCCUGGAAGUCAU 540 1371 GGGAGGGCAUUGUGGCCCG 341 1371GGGAGGGCAUUGUGGCCCG 341 1393 CGGGCCACAAUGCCCUCCC 541 1389GGCUAACAGAGAGCCUGUU 342 1389 GGCUAACAGAGAGCCUGUU 342 1411AACAGGCUCUCUGUUAGCC 542 1407 UCCUGGACCUGCUCGGAGA 343 1407UCCUGGACCUGCUCGGAGA 343 1429 UCUCCGAGCAGGUCCAGGA 543 1425AGGAGAAUGGGGGCUUUUG 344 1425 AGGAGAAUGGGGGCUUUUG 344 1447CAAAAGCCCCCAUUCUCCU 544 1443 GCCAGCAGGACAUGGGGGA 345 1443GCCAGCAGGACAUGGGGGA 345 1465 UCCCCCAUGUCCUGCUGGC 545 1461AGUCAUGCCUUCUUCCACC 346 1461 AGUCAUGCCUUCUUCCACC 346 1483GGUGGAAGAAGGCAUGACU 546 1479 CUUCGGGAAGUACGAGUGC 347 1479CUUCGGGAAGUACGAGUGC 347 1501 GCACUCGUACUUCCCGAAG 547 1497CUCACAUGCCCUGGGAUGA 348 1497 CUCACAUGCCCUGGGAUGA 348 1519UCAUCCCAGGGCAUGUGAG 548 1515 AGUUCCCAAGUGCAGGGCC 349 1515AGUUCCCAAGUGCAGGGCC 349 1537 GGCCCUGCACUUGGGAACU 549 1533CCAAGGAGGCACCUCCCUG 350 1533 CCAAGGAGGCACCUCCCUG 350 1555CAGGGAGGUGCCUCCUUGG 550 1551 GGGGCAAGGAGCAGCCUCU 351 1551GGGGCAAGGAGCAGCCUCU 351 1573 AGAGGCUGCUCCUUGCCCC 551 1569UCCACCUGGAGCCAAGUCC 352 1569 UCCACCUGGAGCCAAGUCC 352 1591GGACUUGGCUCCAGGUGGA 552 1587 CUCCUGCCAGCCCGACCCA 353 1587CUCCUGCCAGCCCGACCCA 353 1609 UGGGUCGGGCUGGCAGGAG 553 1605AGAGUCCAGACAACCUGAC 354 1605 AGAGUCCAGACAACCUGAC 354 1627GUCAGGUUGUCUGGACUCU 554 1623 CUUGCACAGAGACGCCCCU 355 1623CUUGCACAGAGACGCCCCU 355 1645 AGGGGCGUCUCUGUGCAAG 555 1641UCGUCAUCGCAGGCAACCC 356 1641 UCGUCAUCGCAGGCAACCC 356 1663GGGUUGCCUGCGAUGACGA 556 1659 CUGCUUACCGCAGCUUCAG 357 1659CUGCUUACCGCAGCUUCAG 357 1681 CUGAAGCUGCGGUAAGCAG 557 1677GCAACUCCCUGAGCCAGUC 358 1677 GCAACUCCCUGAGCCAGUC 358 1699GACUGGCUCAGGGAGUUGC 558 1695 CACCGUGUCCCAGAGAGCU 359 1695CACCGUGUCCCAGAGAGCU 359 1717 AGCUCUCUGGGACACGGUG 559 1713UGGGUCCAGACCCACUGCU 360 1713 UGGGUCCAGACCCACUGCU 360 1735AGCAGUGGGUCUGGACCCA 560 1731 UGGCCAGACACCUGGAGGA 361 1731UGGCCAGACACCUGGAGGA 361 1753 UCCUCCAGGUGUCUGGCCA 561 1749AAGUAGAACCCGAGAUGCC 362 1749 AAGUAGAACCCGAGAUGCC 362 1771GGCAUCUCGGGUUCUACUU 562 1767 CCUGUGUCCCCCAGCUCUC 363 1767CCUGUGUCCCCCAGCUCUC 363 1789 GAGAGCUGGGGGACACAGG 563 1785CUGAGCCAACCACUGUGCC 364 1785 CUGAGCCAACCACUGUGCC 364 1807GGCACAGUGGUUGGCUCAG 564 1803 CCCAACCUGAGCCAGAAAC 365 1803CCCAACCUGAGCCAGAAAC 365 1825 GUUUCUGGCUCAGGUUGGG 565 1821CCUGGGAGCAGAUCCUCCG 366 1821 CCUGGGAGCAGAUCCUCCG 366 1843CGGAGGAUCUGCUCCCAGG 566 1839 GCCGAAAUGUCCUCCAGCA 367 1839GCCGAAAUGUCCUCCAGCA 367 1861 UGCUGGAGGACAUUUCGGC 567 1857AUGGGGCAGCUGCAGCCCC 368 1857 AUGGGGCAGCUGCAGCCCC 368 1879GGGGCUGCAGCUGCCCCAU 568 1875 CCGUCUCGGCCCCCACCAG 369 1875CCGUCUCGGCCCCCACCAG 369 1897 CUGGUGGGGGCCGAGACGG 569 1893GUGGCUAUCAGGAGUUUGU 370 1893 GUGGCUAUCAGGAGUUUGU 370 1915ACAAACUCCUGAUAGCCAC 570 1911 UACAUGCGGUGGAGCAGGG 371 1911UACAUGCGGUGGAGCAGGG 371 1933 CCCUGCUCCACCGCAUGUA 571 1929GUGGCACCCAGGCCAGUGC 372 1929 GUGGCACCCAGGCCAGUGC 372 1951GCACUGGCCUGGGUGCCAC 572 1947 CGGUGGUGGGCUUGGGUCC 373 1947CGGUGGUGGGCUUGGGUCC 373 1969 GGACCCAAGCCCACCACCG 573 1965CCCCAGGAGAGGCUGGUUA 374 1965 CCCCAGGAGAGGCUGGUUA 374 1987UAACCAGCCUCUCCUGGGG 574 1983 ACAAGGCCUUCUCAAGCCU 375 1983ACAAGGCCUUCUCAAGCCU 375 2005 AGGCUUGAGAAGGCCUUGU 575 2001UGCUUGCCAGCAGUGCUGU 376 2001 UGCUUGCCAGCAGUGCUGU 376 2023ACAGCACUGCUGGCAAGCA 576 2019 UGUCCCCAGAGAAAUGUGG 377 2019UGUCCCCAGAGAAAUGUGG 377 2041 CCACAUUUCUCUGGGGACA 577 2037GGUUUGGGGCUAGCAGUGG 378 2037 GGUUUGGGGCUAGCAGUGG 378 2059CCACUGCUAGCCCCAAACC 578 2055 GGGAAGAGGGGUAUAAGCC 379 2055GGGAAGAGGGGUAUAAGCC 379 2077 GGCUUAUACCCCUCUUCCC 579 2073CUUUCCAAGACCUCAUUCC 380 2073 CUUUCCAAGACCUCAUUCC 380 2095GGAAUGAGGUCUUGGAAAG 580 2091 CUGGCUGCCCUGGGGACCC 381 2091CUGGCUGCCCUGGGGACCC 381 2113 GGGUCCCCAGGGCAGCCAG 581 2109CUGCCCCAGUCCCUGUCCC 382 2109 CUGCCCCAGUCCCUGUCCC 382 2131GGGACAGGGACUGGGGCAG 582 2127 CCUUGUUCACCUUUGGACU 383 2127CCUUGUUCACCUUUGGACU 383 2149 AGUCCAAAGGUGAACAAGG 583 2145UGGACAGGGAGCCACCUCG 384 2145 UGGACAGGGAGCCACCUCG 384 2167CGAGGUGGCUCCCUGUCCA 584 2163 GCAGUCCGCAGAGCUCACA 385 2163GCAGUCCGCAGAGCUCACA 385 2185 UGUGAGCUCUGCGGACUGC 585 2181AUCUCCCAAGCAGCUCCCC 386 2181 AUCUCCCAAGCAGCUCCCC 386 2203GGGGAGCUGCUUGGGAGAU 586 2199 CAGAGCACCUGGGUCUGGA 387 2199CAGAGCACCUGGGUCUGGA 387 2221 UCCAGACCCAGGUGCUCUG 587 2217AGCCGGGGGAAAAGGUAGA 388 2217 AGCCGGGGGAAAAGGUAGA 388 2239UCUACCUUUUCCCCCGGCU 588 2235 AGGACAUGCCAAAGCCCCC 389 2235AGGACAUGCCAAAGCCCCC 389 2257 GGGGGCUUUGGCAUGUCCU 589 2253CACUUCCCCAGGAGCAGGC 390 2253 CACUUCCCCAGGAGCAGGC 390 2275GCCUGCUCCUGGGGAAGUG 590 2271 CCACAGACCCCCUUGUGGA 391 2271CCACAGACCCCCUUGUGGA 391 2293 UCCACAAGGGGGUCUGUGG 591 2289ACAGCCUGGGCAGUGGCAU 392 2289 ACAGCCUGGGCAGUGGCAU 392 2311AUGCCACUGCCCAGGCUGU 592 2307 UUGUCUACUCAGCCCUUAC 393 2307UUGUCUACUCAGCCCUUAC 393 2329 GUAAGGGCUGAGUAGACAA 593 2325CCUGCCACCUGUGCGGCCA 394 2325 CCUGCCACCUGUGCGGCCA 394 2347UGGCCGCACAGGUGGCAGG 594 2343 ACCUGAAACAGUGUCAUGG 395 2343ACCUGAAACAGUGUCAUGG 395 2365 CCAUGACACUGUUUCAGGU 595 2361GCCAGGAGGAUGGUGGCCA 396 2361 GCCAGGAGGAUGGUGGCCA 396 2383UGGCCACCAUCCUCCUGGC 596 2379 AGACCCCUGUCAUGGCCAG 397 2379AGACCCCUGUCAUGGCCAG 397 2401 CUGGCCAUGACAGGGGUCU 597 2397GUCCUUGCUGUGGCUGCUG 398 2397 GUCCUUGCUGUGGCUGCUG 398 2419CAGCAGCCACAGCAAGGAC 598 2415 GCUGUGGAGACAGGUCCUC 399 2415GCUGUGGAGACAGGUCCUC 399 2437 GAGGACCUGUCUCCACAGC 599 2433CGCCCCCUACAACCCCCCU 400 2433 CGCCCCCUACAACCCCCCU 400 2455AGGGGGGUUGUAGGGGGCG 600 2451 UGAGGGCCCCAGACCCCUC 401 2451UGAGGGCCCCAGACCCCUC 401 2473 GAGGGGUCUGGGGCCCUCA 601 2469CUCCAGGUGGGGUUCCACU 402 2469 CUCCAGGUGGGGUUCCACU 402 2491AGUGGAACCCCACCUGGAG 602 2487 UGGAGGCCAGUCUGUGUCC 403 2487UGGAGGCCAGUCUGUGUCC 403 2509 GGACACAGACUGGCCUCCA 603 2505CGGCCUCCCUGGCACCCUC 404 2505 CGGCCUCCCUGGCACCCUC 404 2527GAGGGUGCCAGGGAGGCCG 604 2523 CGGGCAUCUCAGAGAAGAG 405 2523CGGGCAUCUCAGAGAAGAG 405 2545 CUCUUCUCUGAGAUGCCCG 605 2541GUAAAUCCUCAUCAUCCUU 406 2541 GUAAAUCCUCAUCAUCCUU 406 2563AAGGAUGAUGAGGAUUUAC 606 2559 UCCAUCCUGCCCCUGGCAA 407 2559UCCAUCCUGCCCCUGGCAA 407 2581 UUGCCAGGGGCAGGAUGGA 607 2577AUGCUCAGAGCUCAAGCCA 408 2577 AUGCUCAGAGCUCAAGCCA 408 2599UGGCUUGAGCUCUGAGCAU 608 2595 AGACCCCCAAAAUCGUGAA 409 2595AGACCCCCAAAAUCGUGAA 409 2617 UUCACGAUUUUGGGGGUCU 609 2613ACUUUGUCUCCGUGGGACC 410 2613 ACUUUGUCUCCGUGGGACC 410 2635GGUCCCACGGAGACAAAGU 610 2631 CCACAUACAUGAGGGUCUC 411 2631CCACAUACAUGAGGGUCUC 411 2653 GAGACCCUCAUGUAUGUGG 611 2649CUUAGGUGCAUGUCCUCUU 412 2649 CUUAGGUGCAUGUCCUCUU 412 2671AAGAGGACAUGCACCUAAG 612 2667 UGUUGCUGAGUCUGCAGAU 413 2667UGUUGCUGAGUCUGCAGAU 413 2689 AUCUGCAGACUCAGCAACA 613 2685UGAGGACUAGGGCUUAUCC 414 2685 UGAGGACUAGGGCUUAUCC 414 2707GGAUAAGCCCUAGUCCUCA 614 2703 CAUGCCUGGGAAAUGCCAC 415 2703CAUGCCUGGGAAAUGCCAC 415 2725 GUGGCAUUUCCCAGGCAUG 615 2721CCUCCUGGAAGGCAGCCAG 416 2721 CCUCCUGGAAGGCAGCCAG 416 2743CUGGCUGCCUUCCAGGAGG 616 2739 GGCUGGCAGAUUUCCAAAA 417 2739GGCUGGCAGAUUUCCAAAA 417 2761 UUUUGGAAAUCUGCCAGCC 617 2757AGACUUGAAGAACCAUGGU 418 2757 AGACUUGAAGAACCAUGGU 418 2779ACCAUGGUUCUUCAAGUCU 618 2775 UAUGAAGGUGAUUGGCCCC 419 2775UAUGAAGGUGAUUGGCCCC 419 2797 GGGGCCAAUCACCUUCAUA 619 2793CACUGACGUUGGCCUAACA 420 2793 CACUGACGUUGGCCUAACA 420 2815UGUUAGGCCAACGUCAGUG 620 2811 ACUGGGCUGCAGAGACUGG 421 2811ACUGGGCUGCAGAGACUGG 421 2833 CCAGUCUCUGCAGCCCAGU 621 2829GACCCCGCCCAGCAUUGGG 422 2829 GACCCCGCCCAGCAUUGGG 422 2851CCCAAUGCUGGGCGGGGUC 622 2847 GCUGGGCUCGCCACAUCCC 423 2847GCUGGGCUCGCCACAUCCC 423 2869 GGGAUGUGGCGAGCCCAGC 623 2865CAUGAGAGUAGAGGGCACU 424 2865 CAUGAGAGUAGAGGGCACU 424 2887AGUGCCCUCUACUCUCAUG 624 2883 UGGGUCGCCGUGCCCCACG 425 2883UGGGUCGCCGUGCCCCACG 425 2905 CGUGGGGCACGGCGACCCA 625 2901GGCAGGCCCCUGCAGGAAA 426 2901 GGCAGGCCCCUGCAGGAAA 426 2923UUUCCUGCAGGGGCCUGCC 626 2919 AACUGAGGCCCUUGGGCAC 427 2919AACUGAGGCCCUUGGGCAC 427 2941 GUGCCCAAGGGCCUCAGUU 627 2937CCUCGACUUGUGAACGAGU 428 2937 CCUCGACUUGUGAACGAGU 428 2959ACUCGUUCACAAGUCGAGG 628 2955 UUGUUGGCUGCUCCCUCCA 429 2955UUGUUGGCUGCUCCCUCCA 429 2977 UGGAGGGAGCAGCCAACAA 629 2973ACAGCUUCUGCAGCAGACU 430 2973 ACAGCUUCUGCAGCAGACU 430 2995AGUCUGCUGCAGAAGCUGU 630 2991 UGUCCCUGUUGUAACUGCC 431 2991UGUCCCUGUUGUAACUGCC 431 3013 GGCAGUUACAACAGGGACA 631 3009CCAAGGCAUGUUUUGCCCA 432 3009 CCAAGGCAUGUUUUGCCCA 432 3031UGGGCAAAACAUGCCUUGG 632 3027 ACCAGAUCAUGGCCCACGU 433 3027ACCAGAUCAUGGCCCACGU 433 3049 ACGUGGGCCAUGAUCUGGU 633 3045UGGAGGCCCACCUGCCUCU 434 3045 UGGAGGCCCACCUGCCUCU 434 3067AGAGGCAGGUGGGCCUCCA 634 3063 UGUCUCACUGAACUAGAAG 435 3063UGUCUCACUGAACUAGAAG 435 3085 CUUCUAGUUCAGUGAGACA 635 3081GCCGAGCCUAGAAACUAAC 436 3081 GCCGAGCCUAGAAACUAAC 436 3103GUUAGUUUCUAGGCUCGGC 636 3099 CACAGCCAUCAAGGGAAUG 437 3099CACAGCCAUCAAGGGAAUG 437 3121 CAUUCCCUUGAUGGCUGUG 637 3117GACUUGGGCGGCCUUGGGA 438 3117 GACUUGGGCGGCCUUGGGA 438 3139UCCCAAGGCCGCCCAAGUC 638 3135 AAAUCGAUGAGAAAUUGAA 439 3135AAAUCGAUGAGAAAUUGAA 439 3157 UUCAAUUUCUCAUCGAUUU 639 3153ACUUCAGGGAGGGUGGUCA 440 3153 ACUUCAGGGAGGGUGGUCA 440 3175UGACCACCCUCCCUGAAGU 640 3171 AUUGCCUAGAGGUGCUCAU 441 3171AUUGCCUAGAGGUGCUCAU 441 3193 AUGAGCACCUCUAGGCAAU 641 3189UUCAUUUAACAGAGCUUCC 442 3189 UUCAUUUAACAGAGCUUCC 442 3211GGAAGCUCUGUUAAAUGAA 642 3207 CUUAGGUUGAUGCUGGAGG 443 3207CUUAGGUUGAUGCUGGAGG 443 3229 CCUCCAGCAUCAACCUAAG 643 3225GCAGAAUCCCGGCUGUCAA 444 3225 GCAGAAUCCCGGCUGUCAA 444 3247UUGACAGCCGGGAUUCUGC 644 3243 AGGGGUGUUCAGUUAAGGG 445 3243AGGGGUGUUCAGUUAAGGG 445 3265 CCCUUAACUGAACACCCCU 645 3261GGAGCAACAGAGGACAUGA 446 3261 GGAGCAACAGAGGACAUGA 446 3283UCAUGUCCUCUGUUGCUCC 646 3279 AAAAAUUGCUAUGACUAAA 447 3279AAAAAUUGCUAUGACUAAA 447 3301 UUUAGUCAUAGCAAUUUUU 647 3297AGCAGGGACAAUUUGCUGC 448 3297 AGCAGGGACAAUUUGCUGC 448 3319GCAGCAAAUUGUCCCUGCU 648 3315 CCAAACACCCAUGCCCAGC 449 3315CCAAACACCCAUGCCCAGC 449 3337 GCUGGGCAUGGGUGUUUGG 649 3333CUGUAUGGCUGGGGGCUCC 450 3333 CUGUAUGGCUGGGGGCUCC 450 3355GGAGCCCCCAGCCAUACAG 650 3351 CUCGUAUGCAUGGAACCCC 451 3351CUCGUAUGCAUGGAACCCC 451 3373 GGGGUUCCAUGCAUACGAG 651 3369CCAGAAUAAAUAUGCUCAG 452 3369 CCAGAAUAAAUAUGCUCAG 452 3391CUGAGCAUAUUUAUUCUGG 652 3387 GCCACCCUGUGGGCCGGGC 453 3387GCCACCCUGUGGGCCGGGC 453 3409 GCCCGGCCCACAGGGUGGC 653 3405CAAUCCAGACAGCAGGCAU 454 3405 CAAUCCAGACAGCAGGCAU 454 3427AUGCCUGCUGUCUGGAUUG 654 3423 UAAGGCACCAGUUACCCUG 455 3423UAAGGCACCAGUUACCCUG 455 3445 CAGGGUAACUGGUGCCUUA 655 3441GCAUGUUGGCCCAGACCUC 456 3441 GCAUGUUGGCCCAGACCUC 456 3463GAGGUCUGGGCCAACAUGC 656 3459 CAGGUGCUAGGGAAGGCGG 457 3459CAGGUGCUAGGGAAGGCGG 457 3481 CCGCCUUCCCUAGCACCUG 657 3477GGAACCUUGGGUUGAGUAA 458 3477 GGAACCUUGGGUUGAGUAA 458 3499UUACUCAACCCAAGGUUCC 658 3495 AUGCUCGUCUGUGUGUUUU 459 3495AUGCUCGUCUGUGUGUUUU 459 3517 AAAACACACAGACGAGCAU 659 3513UAGUUUCAUCACCUGUUAU 460 3513 UAGUUUCAUCACCUGUUAU 460 3535AUAACAGGUGAUGAAACUA 660 3531 UCUGUGUUUGCUGAGGAGA 461 3531UCUGUGUUUGCUGAGGAGA 461 3553 UCUCCUCAGCAAACACAGA 661 3549AGUGGAACAGAAGGGGUGG 462 3549 AGUGGAACAGAAGGGGUGG 462 3571CCACCCCUUCUGUUCCACU 662 3567 GAGUUUUGUAUAAAUAAAG 463 3567GAGUUUUGUAUAAAUAAAG 463 3589 CUUUAUUUAUACAAAACUC 663 3577UAAAUAAAGUUUCUUUGUC 464 3577 UAAAUAAAGUUUCUUUGUC 464 3599GACAAAGAAACUUUAUUUA 664 IL13 NM_002188 3 GCCACCCAGCCUAUGCAUC 665 3GCCACCCAGCCUAUGCAUC 665 25 GAUGCAUAGGCUGGGUGGC 736 21CCGCUCCUCAAUCCUCUCC 666 21 CCGCUCCUCAAUCCUCUCC 666 43GGAGAGGAUUGAGGAGCGG 737 39 CUGUUGGCACUGGGCCUCA 667 39CUGUUGGCACUGGGCCUCA 667 61 UGAGGCCCAGUGCCAACAG 738 57AUGGCGCUUUUGUUGACCA 668 57 AUGGCGCUUUUGUUGACCA 668 79UGGUCAACAAAAGCGCCAU 739 75 ACGGUCAUUGCUCUCACUU 669 75ACGGUCAUUGCUCUCACUU 669 97 AAGUGAGAGCAAUGACCGU 740 93UGCCUUGGCGGCUUUGCCU 670 93 UGCCUUGGCGGCUUUGCCU 670 115AGGCAAAGCCGCCAAGGCA 741 111 UCCCCAGGCCCUGUGCCUC 671 111UCCCCAGGCCCUGUGCCUC 671 133 GAGGCACAGGGCCUGGGGA 742 129CCCUCUACAGCCCUCAGGG 672 129 CCCUCUACAGCCCUCAGGG 672 151CCCUGAGGGCUGUAGAGGG 743 147 GAGCUCAUUGAGGAGCUGG 673 147GAGCUCAUUGAGGAGCUGG 673 169 CCAGCUCCUCAAUGAGCUC 744 165GUCAACAUCACCCAGAACC 674 165 GUCAACAUCACCCAGAACC 674 187GGUUCUGGGUGAUGUUGAC 745 183 CAGAAGGCUCCGCUCUGCA 675 183CAGAAGGCUCCGCUCUGCA 675 205 UGCAGAGCGGAGCCUUCUG 746 201AAUGGCAGCAUGGUAUGGA 676 201 AAUGGCAGCAUGGUAUGGA 676 223UCCAUACCAUGCUGCCAUU 747 219 AGCAUCAACCUGACAGCUG 677 219AGCAUCAACCUGACAGCUG 677 241 CAGCUGUCAGGUUGAUGCU 748 237GGCAUGUACUGUGCAGCCC 678 237 GGCAUGUACUGUGCAGCCC 678 259GGGCUGCACAGUACAUGCC 749 255 CUGGAAUCCCUGAUCAACG 679 255CUGGAAUCCCUGAUCAACG 679 277 CGUUGAUCAGGGAUUCCAG 750 273GUGUCAGGCUGCAGUGCCA 680 273 GUGUCAGGCUGCAGUGCCA 680 295UGGCACUGCAGCCUGACAC 751 291 AUCGAGAAGACCCAGAGGA 681 291AUCGAGAAGACCCAGAGGA 681 313 UCCUCUGGGUCUUCUCGAU 752 309AUGCUGAGCGGAUUCUGCC 682 309 AUGCUGAGCGGAUUCUGCC 682 331GGCAGAAUCCGCUCAGCAU 753 327 CCGCACAAGGUCUCAGCUG 683 327CCGCACAAGGUCUCAGCUG 683 349 CAGCUGAGACCUUGUGCGG 754 345GGGCAGUUUUCCAGCUUGC 684 345 GGGCAGUUUUCCAGCUUGC 684 367GCAAGCUGGAAAACUGCCC 755 363 CAUGUCCGAGACACCAAAA 685 363CAUGUCCGAGACACCAAAA 685 385 UUUUGGUGUCUCGGACAUG 756 381AUCGAGGUGGCCCAGUUUG 686 381 AUCGAGGUGGCCCAGUUUG 686 403CAAACUGGGCCACCUCGAU 757 399 GUAAAGGACCUGCUCUUAC 687 399GUAAAGGACCUGCUCUUAC 687 421 GUAAGAGCAGGUCCUUUAC 758 417CAUUUAAAGAAACUUUUUC 688 417 CAUUUAAAGAAACUUUUUC 688 439GAAAAAGUUUCUUUAAAUG 759 435 CGCGAGGGACAGUUCAACU 689 435CGCGAGGGACAGUUCAACU 689 457 AGUUGAACUGUCCCUCGCG 760 453UGAAACUUCGAAAGCAUCA 690 453 UGAAACUUCGAAAGCAUCA 690 475UGAUGCUUUCGAAGUUUCA 761 471 AUUAUUUGCAGAGACAGGA 691 471AUUAUUUGCAGAGACAGGA 691 493 UCCUGUCUCUGCAAAUAAU 762 489ACCUGACUAUUGAAGUUGC 692 489 ACCUGACUAUUGAAGUUGC 692 511GCAACUUCAAUAGUCAGGU 763 507 CAGAUUCAUUUUUCUUUCU 693 507CAGAUUCAUUUUUCUUUCU 693 529 AGAAAGAAAAAUGAAUCUG 764 525UGAUGUCAAAAAUGUCUUG 694 525 UGAUGUCAAAAAUGUCUUG 694 547CAAGACAUUUUUGACAUCA 765 543 GGGUAGGCGGGAAGGAGGG 695 543GGGUAGGCGGGAAGGAGGG 695 565 CCCUCCUUCCCGCCUACCC 766 561GUUAGGGAGGGGUAAAAUU 696 561 GUUAGGGAGGGGUAAAAUU 696 583AAUUUUACCCCUCCCUAAC 767 579 UCCUUAGCUUAGACCUCAG 697 579UCCUUAGCUUAGACCUCAG 697 601 CUGAGGUCUAAGCUAAGGA 768 597GCCUGUGCUGCCCGUCUUC 698 597 GCCUGUGCUGCCCGUCUUC 698 619GAAGACGGGCAGCACAGGC 769 615 CAGCCUAGCCGACCUCAGC 699 615CAGCCUAGCCGACCUCAGC 699 637 GCUGAGGUCGGCUAGGCUG 770 633CCUUCCCCUUGCCCAGGGC 700 633 CCUUCCCCUUGCCCAGGGC 700 655GCCCUGGGCAAGGGGAAGG 771 651 CUCAGCCUGGUGGGCCUCC 701 651CUCAGCCUGGUGGGCCUCC 701 673 GGAGGCCCACCAGGCUGAG 772 669CUCUGUCCAGGGCCCUGAG 702 669 CUCUGUCCAGGGCCCUGAG 702 691CUCAGGGCCCUGGACAGAG 773 687 GCUCGGUGGACCCAGGGAU 703 687GCUCGGUGGACCCAGGGAU 703 709 AUCCCUGGGUCCACCGAGC 774 705UGACAUGUCCCUACACCCC 704 705 UGACAUGUCCCUACACCCC 704 727GGGGUGUAGGGACAUGUCA 775 723 CUCCCCUGCCCUAGAGCAC 705 723CUCCCCUGCCCUAGAGCAC 705 745 GUGCUCUAGGGCAGGGGAG 776 741CACUGUAGCAUUACAGUGG 706 741 CACUGUAGCAUUACAGUGG 706 763CCACUGUAAUGCUACAGUG 777 759 GGUGCCCCCCUUGCCAGAC 707 759GGUGCCCCCCUUGCCAGAC 707 781 GUCUGGCAAGGGGGGCACC 778 777CAUGUGGUGGGACAGGGAC 708 777 CAUGUGGUGGGACAGGGAC 708 799GUCCCUGUCCCACCACAUG 779 795 CCCACUUCACACACAGGCA 709 795CCCACUUCACACACAGGCA 709 817 UGCCUGUGUGUGAAGUGGG 780 813AACUGAGGCAGACAGCAGC 710 813 AACUGAGGCAGACAGCAGC 710 835GCUGCUGUCUGCCUCAGUU 781 831 CUCAGGCACACUUCUUCUU 711 831CUCAGGCACACUUCUUCUU 711 853 AAGAAGAAGUGUGCCUGAG 782 849UGGUCUUAUUUAUUAUUGU 712 849 UGGUCUUAUUUAUUAUUGU 712 871ACAAUAAUAAAUAAGACCA 783 867 UGUGUUAUUUAAAUGAGUG 713 867UGUGUUAUUUAAAUGAGUG 713 889 CACUCAUUUAAAUAACACA 784 885GUGUUUGUCACCGUUGGGG 714 885 GUGUUUGUCACCGUUGGGG 714 907CCCCAACGGUGACAAACAC 785 903 GAUUGGGGAAGACUGUGGC 715 903GAUUGGGGAAGACUGUGGC 715 925 GCCACAGUCUUCCCCAAUC 786 921CUGCUAGCACUUGGAGCCA 716 921 CUGCUAGCACUUGGAGCCA 716 943UGGCUCCAAGUGCUAGCAG 787 939 AAGGGUUCAGAGACUCAGG 717 939AAGGGUUCAGAGACUCAGG 717 961 CCUGAGUCUCUGAACCCUU 788 957GGCCCCAGCACUAAAGCAG 718 957 GGCCCCAGCACUAAAGCAG 718 979CUGCUUUAGUGCUGGGGCC 789 975 GUGGACACCAGGAGUCCCU 719 975GUGGACACCAGGAGUCCCU 719 997 AGGGACUCCUGGUGUCCAC 790 933UGGUAAUAAGUACUGUGUA 720 993 UGGUAAUAAGUACUGUGUA 720 1015UACACAGUACUUAUUACCA 791 1011 ACAGAAUUCUGCUACCUCA 721 1011ACAGAAUUCUGCUACCUCA 721 1033 UGAGGUAGCAGAAUUCUGU 792 1029ACUGGGGUCCUGGGGCCUC 722 1029 ACUGGGGUCCUGGGGCCUC 722 1051GAGGCCCCAGGACCCCAGU 793 1047 CGGAGCCUCAUCCGAGGCA 723 1047CGGAGCCUCAUCCGAGGCA 723 1069 UGCCUCGGAUGAGGCUCCG 794 1065AGGGUCAGGAGAGGGGCAG 724 1065 AGGGUCAGGAGAGGGGCAG 724 1087CUGCCCCUCUCCUGACCCU 795 1083 GAACAGCCGCUCCUGUCUG 725 1083GAACAGCCGCUCCUGUCUG 725 1105 CAGACAGGAGCGGCUGUUC 796 1101GCCAGCCAGCAGCCAGCUC 726 1101 GCCAGCCAGCAGCCAGCUC 726 1123GAGCUGGCUGCUGGCUGGC 797 1119 CUCAGCCAACGAGUAAUUU 727 1119CUCAGCCAACGAGUAAUUU 727 1141 AAAUUACUCGUUGGCUGAG 798 1137UAUUGUUUUUCCUUGUAUU 728 1137 UAUUGUUUUUCCUUGUAUU 728 1159AAUACAAGGAAAAACAAUA 799 1155 UUAAAUAUUAAAUAUGUUA 729 1155UUAAAUAUUAAAUAUGUUA 729 1177 UAACAUAUUUAAUAUUUAA 800 1173AGCAAAGAGUUAAUAUAUA 730 1173 AGCAAAGAGUUAAUAUAUA 730 1195UAUAUAUUAACUCUUUGCU 801 1191 AGAAGGGUACCUUGAACAC 731 1191AGAAGGGUACCUUGAACAC 731 1213 GUGUUCAAGGUACCCUUCU 802 1209CUGGGGGAGGGGACAUUGA 732 1209 CUGGGGGAGGGGACAUUGA 732 1231UCAAUGUCCCCUCCCCCAG 803 1227 AACAAGUUGUUUCAUUGAC 733 1227AACAAGUUGUUUCAUUGAC 733 1249 GUCAAUGAAACAACUUGUU 804 1245CUAUCAAACUGAAGCCAGA 734 1245 CUAUCAAACUGAAGCCAGA 734 1267UCUGGCUUCAGUUUGAUAG 805 1262 GAAAUAAAGUUGGUGACAG 735 1262GAAAUAAAGUUGGUGACAG 735 1284 CUGUCACCAACUUUAUUUC 806 IL13RA1 NM_001560 3CCAAGGCUCCAGCCCGGCC 807 3 CCAAGGCUCCAGCCCGGCC 807 25 GGCCGGGCUGGAGCCUUGG1030 21 CGGGCUCCGAGGCGAGAGG 808 21 CGGGCUCCGAGGCGAGAGG 808 43CCUCUCGCCUCGGAGCCCG 1031 39 GCUGCAUGGAGUGGCCGGC 809 39GCUGCAUGGAGUGGCCGGC 809 61 GCCGGCCACUCCAUGCAGC 1032 57CGCGGCUCUGCGGGCUGUG 810 57 CGCGGCUCUGCGGGCUGUG 810 79CACAGCCCGCAGAGCCGCG 1033 75 GGGCGCUGCUGCUCUGCGC 811 75GGGCGCUGCUGCUCUGCGC 811 97 GCGCAGAGCAGCAGCGCCC 1034 93CCGGCGGCGGGGGCGGGGG 812 93 CCGGCGGCGGGGGCGGGGG 812 115CCCCCGCCCCCGCCGCCGG 1035 111 GCGGGGGCGCCGCGCCUAC 813 111GCGGGGGCGCCGCGCCUAC 813 133 GUAGGCGCGGCGCCCCCGC 1036 129CGGAAACUCAGCCACCUGU 814 129 CGGAAACUCAGCCACCUGU 814 151ACAGGUGGCUGAGUUUCCG 1037 147 UGACAAAUUUGAGUGUCUC 815 147UGACAAAUUUGAGUGUCUC 815 169 GAGACACUCAAAUUUGUCA 1038 165CUGUUGAAAACCUCUGCAC 816 165 CUGUUGAAAACCUCUGCAC 816 187GUGCAGAGGUUUUCAACAG 1039 183 CAGUAAUAUGGACAUGGAA 817 183CAGUAAUAUGGACAUGGAA 817 205 UUCCAUGUCCAUAUUACUG 1040 201AUCCACCCGAGGGAGCCAG 818 201 AUCCACCCGAGGGAGCCAG 818 223CUGGCUCCCUCGGGUGGAU 1041 219 GCUCAAAUUGUAGUCUAUG 819 219GCUCAAAUUGUAGUCUAUG 819 241 CAUAGACUACAAUUUGAGC 1042 237GGUAUUUUAGUCAUUUUGG 820 237 GGUAUUUUAGUCAUUUUGG 820 259CCAAAAUGACUAAAAUACC 1043 255 GCGACAAACAAGAUAAGAA 821 255GCGACAAACAAGAUAAGAA 821 277 UUCUUAUCUUGUUUGUCGC 1044 273AAAUAGCUCCGGAAACUCG 822 273 AAAUAGCUCCGGAAACUCG 822 295CGAGUUUCCGGAGCUAUUU 1045 291 GUCGUUCAAUAGAAGUACC 823 291GUCGUUCAAUAGAAGUACC 823 313 GGUACUUCUAUUGAACGAC 1046 309CCCUGAAUGAGAGGAUUUG 824 309 CCCUGAAUGAGAGGAUUUG 824 331CAAAUCCUCUCAUUCAGGG 1047 327 GUCUGCAAGUGGGGUCCCA 825 327GUCUGCAAGUGGGGUCCCA 825 349 UGGGACCCCACUUGCAGAC 1048 345AGUGUAGCACCAAUGAGAG 826 345 AGUGUAGCACCAAUGAGAG 826 367CUCUCAUUGGUGCUACACU 1049 363 GUGAGAAGCCUAGCAUUUU 827 363GUGAGAAGCCUAGCAUUUU 827 385 AAAAUGCUAGGCUUCUCAC 1050 381UGGUUGAAAAAUGCAUCUC 828 381 UGGUUGAAAAAUGCAUCUC 828 403GAGAUGCAUUUUUCAACCA 1051 399 CACCCCCAGAAGGUGAUCC 829 399CACCCCCAGAAGGUGAUCC 829 421 GGAUCACCUUCUGGGGGUG 1052 417CUGAGUCUGCUGUGACUGA 830 417 CUGAGUCUGCUGUGACUGA 830 439UCAGUCACAGCAGACUCAG 1053 435 AGCUUCAAUGCAUUUGGCA 831 435AGCUUCAAUGCAUUUGGCA 831 457 UGCCAAAUGCAUUGAAGCU 1054 453ACAACCUGAGCUACAUGAA 832 453 ACAACCUGAGCUACAUGAA 832 475UUCAUGUAGCUCAGGUUGU 1055 471 AGUGUUCUUGGCUCCCUGG 833 471AGUGUUCUUGGCUCCCUGG 833 493 CCAGGGAGCCAAGAACACU 1056 489GAAGGAAUACCAGUCCCGA 834 489 GAAGGAAUACCAGUCCCGA 834 511UCGGGACUGGUAUUCCUUC 1057 507 ACACUAACUAUACUCUCUA 835 507ACACUAACUAUACUCUCUA 835 529 UAGAGAGUAUAGUUAGUGU 1058 525ACUAUUGGCACAGAAGCCU 836 525 ACUAUUGGCACAGAAGCCU 836 547AGGCUUCUGUGCCAAUAGU 1059 543 UGGAAAAAAUUCAUCAAUG 837 543UGGAAAAAAUUCAUCAAUG 837 565 CAUUGAUGAAUUUUUUCCA 1060 561GUGAAAACAUCUUUAGAGA 838 561 GUGAAAACAUCUUUAGAGA 838 583UCUCUAAAGAUGUUUUCAC 1061 579 AAGGCCAAUACUUUGGUUG 839 579AAGGCCAAUACUUUGGUUG 839 601 CAACCAAAGUAUUGGCCUU 1062 597GUUCCUUUGAUCUGACCAA 840 597 GUUCCUUUGAUCUGACCAA 840 619UUGGUCAGAUCAAAGGAAC 1063 615 AAGUGAAGGAUUCCAGUUU 841 615AAGUGAAGGAUUCCAGUUU 841 637 AAACUGGAAUCCUUCACUU 1064 633UUGAACAACACAGUGUCCA 842 633 UUGAACAACACAGUGUCCA 842 655UGGACACUGUGUUGUUCAA 1065 651 AAAUAAUGGUCAAGGAUAA 843 651AAAUAAUGGUCAAGGAUAA 843 673 UUAUCCUUGACCAUUAUUU 1066 669AUGCAGGAAAAAUUAAACC 844 669 AUGCAGGAAAAAUUAAACC 844 691GGUUUAAUUUUUCCUGCAU 1067 687 CAUCCUUCAAUAUAGUGCC 845 687CAUCCUUCAAUAUAGUGCC 845 709 GGCACUAUAUUGAAGGAUG 1068 705CUUUAACUUCCCGUGUGAA 846 705 CUUUAACUUCCCGUGUGAA 846 727UUCACACGGGAAGUUAAAG 1069 723 AACCUGAUCCUCCACAUAU 847 723AACCUGAUCCUCCACAUAU 847 745 AUAUGUGGAGGAUCAGGUU 1070 741UUAAAAACCUCUCCUUCCA 848 741 UUAAAAACCUCUCCUUCCA 848 763UGGAAGGAGAGGUUUUUAA 1071 759 ACAAUGAUGACCUAUAUGU 849 759ACAAUGAUGACCUAUAUGU 849 781 ACAUAUAGGUCAUCAUUGU 1072 777UGCAAUGGGAGAAUCCACA 850 777 UGCAAUGGGAGAAUCCACA 850 799UGUGGAUUCUCCCAUUGCA 1073 795 AGAAUUUUAUUAGCAGAUG 851 795AGAAUUUUAUUAGCAGAUG 851 817 CAUCUGCUAAUAAAAUUCU 1074 813GCCUAUUUUAUGAAGUAGA 852 813 GCCUAUUUUAUGAAGUAGA 852 835UCUACUUCAUAAAAUAGGC 1075 831 AAGUCAAUAACAGCCAAAC 853 831AAGUCAAUAACAGCCAAAC 853 853 GUUUGGCUGUUAUUGACUU 1076 849CUGAGACACAUAAUGUUUU 854 849 CUGAGACACAUAAUGUUUU 854 871AAAACAUUAUGUGUCUCAG 1077 867 UCUACGUCCAAGAGGCUAA 855 867UCUACGUCCAAGAGGCUAA 855 889 UUAGCCUCUUGGACGUAGA 1078 885AAUGUGAGAAUCCAGAAUU 856 885 AAUGUGAGAAUCCAGAAUU 856 907AAUUCUGGAUUCUCACAUU 1079 903 UUGAGAGAAAUGUGGAGAA 857 903UUGAGAGAAAUGUGGAGAA 857 925 UUCUCCACAUUUCUCUCAA 1080 921AUACAUCUUGUUUCAUGGU 858 921 AUACAUCUUGUUUCAUGGU 858 943ACCAUGAAACAAGAUGUAU 1081 939 UCCCUGGUGUUCUUCCUGA 859 939UCCCUGGUGUUCUUCCUGA 859 961 UCAGGAAGAACACCAGGGA 1082 957AUACUUUGAACACAGUCAG 860 957 AUACUUUGAACACAGUCAG 860 979CUGACUGUGUUCAAAGUAU 1083 975 GAAUAAGAGUCAAAACAAA 861 975GAAUAAGAGUCAAAACAAA 861 997 UUUGUUUUGACUCUUAUUC 1084 993AUAAGUUAUGCUAUGAGGA 862 993 AUAAGUUAUGCUAUGAGGA 862 1015UCCUCAUAGCAUAACUUAU 1085 1011 AUGACAAACUCUGGAGUAA 863 1011AUGACAAACUCUGGAGUAA 863 1033 UUACUCCAGAGUUUGUCAU 1086 1029AUUGGAGCCAAGAAAUGAG 864 1029 AUUGGAGCCAAGAAAUGAG 864 1051CUCAUUUCUUGGCUCCAAU 1087 1047 GUAUAGGUAAGAAGCGCAA 865 1047GUAUAGGUAAGAAGCGCAA 865 1069 UUGCGCUUCUUACCUAUAC 1088 1065AUUCCACACUCUACAUAAC 866 1065 AUUCCACACUCUACAUAAC 866 1087GUUAUGUAGAGUGUGGAAU 1089 1083 CCAUGUUACUCAUUGUUCC 867 1083CCAUGUUACUCAUUGUUCC 867 1105 GGAACAAUGAGUAACAUGG 1090 1101CAGUCAUCGUCGCAGGUGC 868 1101 CAGUCAUCGUCGCAGGUGC 868 1123GCACCUGCGACGAUGACUG 1091 1119 CAAUCAUAGUACUCCUGCU 869 1119CAAUCAUAGUACUCCUGCU 869 1141 AGCAGGAGUACUAUGAUUG 1092 1137UUUACCUAAAAAGGCUCAA 870 1137 UUUACCUAAAAAGGCUCAA 870 1159UUGAGCCUUUUUAGGUAAA 1093 1155 AGAUUAUUAUAUUCCCUCC 871 1155AGAUUAUUAUAUUCCCUCC 871 1177 GGAGGGAAUAUAAUAAUCU 1094 1173CAAUUCCUGAUCCUGGCAA 872 1173 CAAUUCCUGAUCCUGGCAA 872 1195UUGCCAGGAUCAGGAAUUG 1095 1191 AGAUUUUUAAAGAAAUGUU 873 1191AGAUUUUUAAAGAAAUGUU 873 1213 AACAUUUCUUUAAAAAUCU 1096 1209UUGGAGACCAGAAUGAUGA 874 1209 UUGGAGACCAGAAUGAUGA 874 1231UCAUCAUUCUGGUCUCCAA 1097 1227 AUACUCUGCACUGGAAGAA 875 1227AUACUCUGCACUGGAAGAA 875 1249 UUCUUCCAGUGCAGAGUAU 1098 1245AGUACGACAUCUAUGAGAA 876 1245 AGUACGACAUCUAUGAGAA 876 1267UUCUCAUAGAUGUCGUACU 1099 1263 AGCAAACCAAGGAGGAAAC 877 1263AGCAAACCAAGGAGGAAAC 877 1285 GUUUCCUCCUUGGUUUGCU 1100 1281CCGACUCUGUAGUGCUGAU 878 1281 CCGACUCUGUAGUGCUGAU 878 1303AUCAGCACUACAGAGUCGG 1101 1299 UAGAAAACCUGAAGAAAGC 879 1299UAGAAAACCUGAAGAAAGC 879 1321 GCUUUCUUCAGGUUUUCUA 1102 1317CCUCUCAGUGAUGGAGAUA 880 1317 CCUCUCAGUGAUGGAGAUA 880 1339UAUCUCCAUCACUGAGAGG 1103 1335 AAUUUAUUUUUACCUUCAC 881 1335AAUUUAUUUUUACCUUCAC 881 1357 GUGAAGGUAAAAAUAAAUU 1104 1353CUGUGACCUUGAGAAGAUU 882 1353 CUGUGACCUUGAGAAGAUU 882 1375AAUCUUCUCAAGGUCACAG 1105 1371 UCUUCCCAUUCUCCAUUUG 883 1371UCUUCCCAUUCUCCAUUUG 883 1393 CAAAUGGAGAAUGGGAAGA 1106 1389GUUAUCUGGGAACUUAUUA 884 1389 GUUAUCUGGGAACUUAUUA 884 1411UAAUAAGUUCCCAGAUAAC 1107 1407 AAAUGGAAACUGAAACUAC 885 1407AAAUGGAAACUGAAACUAC 885 1429 GUAGUUUCAGUUUCCAUUU 1108 1425CUGCACCAUUUAAAAACAG 886 1425 CUGCACCAUUUAAAAACAG 886 1447CUGUUUUUAAAUGGUGCAG 1109 1443 GGCAGCUCAUAAGAGCCAC 887 1443GGCAGCUCAUAAGAGCCAC 887 1465 GUGGCUCUUAUGAGCUGCC 1110 1461CAGGUCUUUAUGUUGAGUC 888 1461 CAGGUCUUUAUGUUGAGUC 888 1483GACUCAACAUAAAGACCUG 1111 1479 CGCGCACCGAAAAACUAAA 889 1479CGCGCACCGAAAAACUAAA 889 1501 UUUAGUUUUUCGGUGCGCG 1112 1497AAAUAAUGGGCGCUUUGGA 890 1497 AAAUAAUGGGCGCUUUGGA 890 1519UCCAAAGCGCCCAUUAUUU 1113 1515 AGAAGAGUGUGGAGUCAUU 891 1515AGAAGAGUGUGGAGUCAUU 891 1537 AAUGACUCCACACUCUUCU 1114 1533UCUCAUUGAAUUAUAAAAG 892 1533 UCUCAUUGAAUUAUAAAAG 892 1555CUUUUAUAAUUCAAUGAGA 1115 1551 GCCAGCAGGCUUCAAACUA 893 1551GCCAGCAGGCUUCAAACUA 893 1573 UAGUUUGAAGCCUGCUGGC 1116 1569AGGGGACAAAGCAAAAAGU 894 1569 AGGGGACAAAGCAAAAAGU 894 1591ACUUUUUGCUUUGUCCCCU 1117 1587 UGAUGAUAGUGGUGGAGUU 895 1587UGAUGAUAGUGGUGGAGUU 895 1609 AACUCCACCACUAUCAUCA 1118 1605UAAUCUUAUCAAGAGUUGU 896 1605 UAAUCUUAUCAAGAGUUGU 896 1627ACAACUCUUGAUAAGAUUA 1119 1623 UGACAACUUCCUGAGGGAU 897 1623UGACAACUUCCUGAGGGAU 897 1645 AUCCCUCAGGAAGUUGUCA 1120 1641UCUAUACUUGCUUUGUGUU 898 1641 UCUAUACUUGCUUUGUGUU 898 1663AACACAAAGCAAGUAUAGA 1121 1659 UCUUUGUGUCAACAUGAAC 899 1659UCUUUGUGUCAACAUGAAC 899 1681 GUUCAUGUUGACACAAAGA 1122 1677CAAAUUUUAUUUGUAGGGG 900 1677 CAAAUUUUAUUUGUAGGGG 900 1699CCCCUACAAAUAAAAUUUG 1123 1695 GAACUCAUUUGGGGUGCAA 901 1695GAACUCAUUUGGGGUGCAA 901 1717 UUGCACCCCAAAUGAGUUC 1124 1713AAUGCUAAUGUCAAACUUG 902 1713 AAUGCUAAUGUCAAACUUG 902 1735CAAGUUUGACAUUAGCAUU 1125 1731 GAGUCACAAAGAACAUGUA 903 1731GAGUCACAAAGAACAUGUA 903 1753 UACAUGUUCUUUGUGACUC 1126 1749AGAAAACAAAAUGGAUAAA 904 1749 AGAAAACAAAAUGGAUAAA 904 1771UUUAUCCAUUUUGUUUUCU 1127 1767 AAUCUGAUAUGUAUUGUUU 905 1767AAUCUGAUAUGUAUUGUUU 905 1789 AAACAAUACAUAUCAGAUU 1128 1785UGGGAUCCUAUUGAACCAU 906 1785 UGGGAUCCUAUUGAACCAU 906 1807AUGGUUCAAUAGGAUCCCA 1129 1803 UGUUUGUGGCUAUUAAAAC 907 1803UGUUUGUGGCUAUUAAAAC 907 1825 GUUUUAAUAGCCACAAACA 1130 1821CUCUUUUAACAGUCUGGGC 908 1821 CUCUUUUAACAGUCUGGGC 908 1843GCCCAGACUGUUAAAAGAG 1131 1839 CUGGGUCCGGUGGCUCACG 909 1839CUGGGUCCGGUGGCUCACG 909 1861 CGUGAGCCACCGGACCCAG 1132 1857GCCUGUAAUCCCAGCAAUU 910 1857 GCCUGUAAUCCCAGCAAUU 910 1879AAUUGCUGGGAUUACAGGC 1133 1875 UUGGGAGUCCGAGGCGGGC 911 1875UUGGGAGUCCGAGGCGGGC 911 1897 GCCCGCCUCGGACUCCCAA 1134 1893CGGAUCACUCGAGGUCAGG 912 1893 CGGAUCACUCGAGGUCAGG 912 1915CCUGACCUCGAGUGAUCCG 1135 1911 GAGUUCCAGACCAGCCUGA 913 1911GAGUUCCAGACCAGCCUGA 913 1933 UCAGGCUGGUCUGGAACUC 1136 1929ACCAAAAUGGUGAAACCUC 914 1929 ACCAAAAUGGUGAAACCUC 914 1951GAGGUUUCACCAUUUUGGU 1137 1947 CCUCUCUACUAAAACUACA 915 1947CCUCUCUACUAAAACUACA 915 1969 UGUAGUUUUAGUAGAGAGG 1138 1965AAAAAUUAACUGGGUGUGG 916 1965 AAAAAUUAACUGGGUGUGG 916 1987CCACACCCAGUUAAUUUUU 1139 1983 GUGGCGCGUGCCUGUAAUC 917 1983GUGGCGCGUGCCUGUAAUC 917 2005 GAUUACAGGCACGCGCCAC 1140 2001CCCAGCUACUCGGGAAGCU 918 2001 CCCAGCUACUCGGGAAGCU 918 2023AGCUUCCCGAGUAGCUGGG 1141 2019 UGAGGCAGGUGAAUUGUUU 919 2019UGAGGCAGGUGAAUUGUUU 919 2041 AAACAAUUCACCUGCCUCA 1142 2037UGAACCUGGGAGGUGGAGG 920 2037 UGAACCUGGGAGGUGGAGG 920 2059CCUCCACCUCCCAGGUUCA 1143 2055 GUUGCAGUGAGCAGAGAUC 921 2055GUUGCAGUGAGCAGAGAUC 921 2077 GAUCUCUGCUCACUGCAAC 1144 2073CACACCACUGCACUCUAGC 922 2073 CACACCACUGCACUCUAGC 922 2095GCUAGAGUGCAGUGGUGUG 1145 2091 CCUGGGUGACAGAGCAAGA 923 2091CCUGGGUGACAGAGCAAGA 923 2113 UCUUGCUCUGUCACCCAGG 1146 2109ACUCUGUCUAAAAAACAAA 924 2109 ACUCUGUCUAAAAAACAAA 924 2131UUUGUUUUUUAGACAGAGU 1147 2127 AACAAAACAAAACAAAACA 925 2127AACAAAACAAAACAAAACA 925 2149 UGUUUUGUUUUGUUUUGUU 1148 2145AAAAAAACCUCUUAAUAUU 926 2145 AAAAAAACCUCUUAAUAUU 926 2167AAUAUUAAGAGGUUUUUUU 1149 2163 UCUGGAGUCAUCAUUCCCU 927 2163UCUGGAGUCAUCAUUCCCU 927 2185 AGGGAAUGAUGACUCCAGA 1150 2181UUCGACAGCAUUUUCCUCU 928 2181 UUCGACAGCAUUUUCCUCU 928 2203AGAGGAAAAUGCUGUCGAA 1151 2199 UGCUUUGAAAGCCCCAGAA 929 2199UGCUUUGAAAGCCCCAGAA 929 2221 UUCUGGGGCUUUCAAAGCA 1152 2217AAUCAGUGUUGGCCAUGAU 930 2217 AAUCAGUGUUGGCCAUGAU 930 2239AUCAUGGCCAACACUGAUU 1153 2235 UGACAACUACAGAAAAACC 931 2235UGACAACUACAGAAAAACC 931 2257 GGUUUUUCUGUAGUUGUCA 1154 2253CAGAGGCAGCUUCUUUGCC 932 2253 CAGAGGCAGCUUCUUUGCC 932 2275GGCAAAGAAGCUGCCUCUG 1155 2271 CAAGACCUUUCAAAGCCAU 933 2271CAAGACCUUUCAAAGCCAU 933 2293 AUGGCUUUGAAAGGUCUUG 1156 2289UUUUAGGCUGUUAGGGGCA 934 2289 UUUUAGGCUGUUAGGGGCA 934 2311UGCCCCUAACAGCCUAAAA 1157 2307 AGUGGAGGUAGAAUGACUC 935 2307AGUGGAGGUAGAAUGACUC 935 2329 GAGUCAUUCUACCUCCACU 1158 2325CCUUGGGUAUUAGAGUUUC 936 2325 CCUUGGGUAUUAGAGUUUC 936 2347GAAACUCUAAUACCCAAGG 1159 2343 CAACCAUGAAGUCUCUAAC 937 2343CAACCAUGAAGUCUCUAAC 937 2365 GUUAGAGACUUCAUGGUUG 1160 2361CAAUGUAUUUUCUUCACCU 938 2361 CAAUGUAUUUUCUUCACCU 938 2383AGGUGAAGAAAAUACAUUG 1161 2379 UCUGCUACUCAAGUAGCAU 939 2379UCUGCUACUCAAGUAGCAU 939 2401 AUGCUACUUGAGUAGCAGA 1162 2397UUUACUGUGUCUUUGGUUU 940 2397 UUUACUGUGUCUUUGGUUU 940 2419AAACCAAAGACACAGUAAA 1163 2415 UGUGCUAGGCCCCCGGGUG 941 2415UGUGCUAGGCCCCCGGGUG 941 2437 CACCCGGGGGCCUAGCACA 1164 2433GUGAAGCACAGACCCCUUC 942 2433 GUGAAGCACAGACCCCUUC 942 2455GAAGGGGUCUGUGCUUCAC 1165 2451 CCAGGGGUUUACAGUCUAU 943 2451CCAGGGGUUUACAGUCUAU 943 2473 AUAGACUGUAAACCCCUGG 1166 2469UUUGAGACUCCUCAGUUCU 944 2469 UUUGAGACUCCUCAGUUCU 944 2491AGAACUGAGGAGUCUCAAA 1167 2487 UUGCCACUUUUUUUUUUAA 945 2487UUGCCACUUUUUUUUUUAA 945 2509 UUAAAAAAAAAAGUGGCAA 1168 2505AUCUCCACCAGUCAUUUUU 946 2505 AUCUCCACCAGUCAUUUUU 946 2527AAAAAUGACUGGUGGAGAU 1169 2523 UCAGACCUUUUAACUCCUC 947 2523UCAGACCUUUUAACUCCUC 947 2545 GAGGAGUUAAAAGGUCUGA 1170 2541CAAUUCCAACACUGAUUUC 948 2541 CAAUUCCAACACUGAUUUC 948 2563GAAAUCAGUGUUGGAAUUG 1171 2559 CCCCUUUUGCAUUCUCCCU 949 2559CCCCUUUUGCAUUCUCCCU 949 2581 AGGGAGAAUGCAAAAGGGG 1172 2577UCCUUCCCUUCCUUGUAGC 950 2577 UCCUUCCCUUCCUUGUAGC 950 2599GCUACAAGGAAGGGAAGGA 1173 2595 CCUUUUGACUUUCAUUGGA 951 2595CCUUUUGACUUUCAUUGGA 951 2617 UCCAAUGAAAGUCAAAAGG 1174 2613AAAUUAGGAUGUAAAUCUG 952 2613 AAAUUAGGAUGUAAAUCUG 952 2635CAGAUUUACAUCCUAAUUU 1175 2631 GCUCAGGAGACCUGGAGGA 953 2631GCUCAGGAGACCUGGAGGA 953 2653 UCCUCCAGGUCUCCUGAGC 1176 2649AGCAGAGGAUAAUUAGCAU 954 2649 AGCAGAGGAUAAUUAGCAU 954 2671AUGCUAAUUAUCCUCUGCU 1177 2667 UCUCAGGUUAAGUGUGAGU 955 2667UCUCAGGUUAAGUGUGAGU 955 2689 ACUCACACUUAACCUGAGA 1178 2685UAAUCUGAGAAACAAUGAC 956 2685 UAAUCUGAGAAACAAUGAC 956 2707GUCAUUGUUUCUCAGAUUA 1179 2703 CUAAUUCUUGCAUAUUUUG 957 2703CUAAUUCUUGCAUAUUUUG 957 2725 CAAAAUAUGCAAGAAUUAG 1180 2721GUAACUUCCAUGUGAGGGU 958 2721 GUAACUUCCAUGUGAGGGU 958 2743ACCCUCACAUGGAAGUUAC 1181 2739 UUUUCAGCAUUGAUAUUUG 959 2739UUUUCAGCAUUGAUAUUUG 959 2761 CAAAUAUCAAUGCUGAAAA 1182 2757GUGCAUUUUCUAAACAGAG 960 2757 GUGCAUUUUCUAAACAGAG 960 2779CUCUGUUUAGAAAAUGCAC 1183 2775 GAUGAGGUGGUAUCUUCAC 961 2775GAUGAGGUGGUAUCUUCAC 961 2797 GUGAAGAUACCACCUCAUC 1184 2793CGUAGAACAUUGGUAUUCG 962 2793 CGUAGAACAUUGGUAUUCG 962 2815CGAAUACCAAUGUUCUACG 1185 2811 GCUUGAGAAAAAAAGAAUA 963 2811GCUUGAGAAAAAAAGAAUA 963 2833 UAUUCUUUUUUUCUCAAGC 1186 2829AGUUGAACCUAUUUCUCUU 964 2829 AGUUGAACCUAUUUCUCUU 964 2851AAGAGAAAUAGGUUCAACU 1187 2847 UUCUUUACAAGAUGGGUCC 965 2847UUCUUUACAAGAUGGGUCC 965 2869 GGACCCAUCUUGUAAAGAA 1188 2865CAGGAUUCCUCUUUUCUCU 966 2865 CAGGAUUCCUCUUUUCUCU 966 2887AGAGAAAAGAGGAAUCCUG 1189 2883 UGCCAUAAAUGAUUAAUUA 967 2883UGCCAUAAAUGAUUAAUUA 967 2905 UAAUUAAUCAUUUAUGGCA 1190 2901AAAUAGCUUUUGUGUCUUA 968 2901 AAAUAGCUUUUGUGUCUUA 968 2923UAAGACACAAAAGCUAUUU 1191 2919 ACAUUGGUAGCCAGCCAGC 969 2919ACAUUGGUAGCCAGCCAGC 969 2941 GCUGGCUGGCUACCAAUGU 1192 2937CCAAGGCUCUGUUUAUGCU 970 2937 CCAAGGCUCUGUUUAUGCU 970 2959AGCAUAAACAGAGCCUUGG 1193 2955 UUUUGGGGGGCAUAUAUUG 971 2955UUUUGGGGGGCAUAUAUUG 971 2977 CAAUAUAUGCCCCCCAAAA 1194 2973GGGUUCCAUUCUCACCUAU 972 2973 GGGUUCCAUUCUCACCUAU 972 2995AUAGGUGAGAAUGGAACCC 1195 2991 UCCACACAACAUAUCCGUA 973 2991UCCACACAACAUAUCCGUA 973 3013 UACGGAUAUGUUGUGUGGA 1196 3009AUAUAUCCCCUCUACUCUU 974 3009 AUAUAUCCCCUCUACUCUU 974 3031AAGAGUAGAGGGGAUAUAU 1197 3027 UACUUCCCCCAAAUUUAAA 975 3027UACUUCCCCCAAAUUUAAA 975 3049 UUUAAAUUUGGGGGAAGUA 1198 3045AGAAGUAUGGGAAAUGAGA 976 3045 AGAAGUAUGGGAAAUGAGA 976 3067UCUCAUUUCCCAUACUUCU 1199 3063 AGGCAUUUCCCCCACCCCA 977 3063AGGCAUUUCCCCCACCCCA 977 3085 UGGGGUGGGGGAAAUGCCU 1200 3081AUUUCUCUCCUCACACACA 978 3081 AUUUCUCUCCUCACACACA 978 3103UGUGUGUGAGGAGAGAAAU 1201 3099 AGACUCAUAUUACUGGUAG 979 3099AGACUCAUAUUACUGGUAG 979 3121 CUACCAGUAAUAUGAGUCU 1202 3117GGAACUUGAGAACUUUAUU 980 3117 GGAACUUGAGAACUUUAUU 980 3139AAUAAAGUUCUCAAGUUCC 1203 3135 UUCCAAGUUGUUCAAACAU 981 3135UUCCAAGUUGUUCAAACAU 981 3157 AUGUUUGAACAACUUGGAA 1204 3153UUUACCAAUCAUAUUAAUA 982 3153 UUUACCAAUCAUAUUAAUA 982 3175UAUUAAUAUGAUUGGUAAA 1205 3171 ACAAUGAUGCUAUUUGCAA 983 3171ACAAUGAUGCUAUUUGCAA 983 3193 UUGCAAAUAGCAUCAUUGU 1206 3189AUUCCUGCUCCUAGGGGAG 984 3189 AUUCCUGCUCCUAGGGGAG 984 3211CUCCCCUAGGAGCAGGAAU 1207 3207 GGGGAGAUAAGAAACCCUC 985 3207GGGGAGAUAAGAAACCCUC 985 3229 GAGGGUUUCUUAUCUCCCC 1208 3225CACUCUCUACAGGUUUGGG 986 3225 CACUCUCUACAGGUUUGGG 986 3247CCCAAACCUGUAGAGAGUG 1209 3243 GUACAAGUGGCAACCUGCU 987 3243GUACAAGUGGCAACCUGCU 987 3265 AGCAGGUUGCCACUUGUAC 1210 3261UUCCAUGGCCGUGUAGAAG 988 3261 UUCCAUGGCCGUGUAGAAG 988 3283CUUCUACACGGCCAUGGAA 1211 3279 GCAUGGUGCCCUGGCUUCU 989 3279GCAUGGUGCCCUGGCUUCU 989 3301 AGAAGCCAGGGCACCAUGC 1212 3297UCUGAGGAAGCUGGGGUUC 990 3297 UCUGAGGAAGCUGGGGUUC 990 3319GAACCCCAGCUUCCUCAGA 1213 3315 CAUGACAAUGGCAGAUGUA 991 3315CAUGACAAUGGCAGAUGUA 991 3337 UACAUCUGCCAUUGUCAUG 1214 3333AAAGUUAUUCUUGAAGUCA 992 3333 AAAGUUAUUCUUGAAGUCA 992 3355UGACUUCAAGAAUAACUUU 1215 3351 AGAUUGAGGCUGGGAGACA 993 3351AGAUUGAGGCUGGGAGACA 993 3373 UGUCUCCCAGCCUCAAUCU 1216 3369AGCCGUAGUAGAUGUUCUA 994 3369 AGCCGUAGUAGAUGUUCUA 994 3391UAGAACAUCUACUACGGCU 1217 3387 ACUUUGUUCUGCUGUUCUC 995 3387ACUUUGUUCUGCUGUUCUC 995 3409 GAGAACAGCAGAACAAAGU 1218 3405CUAGAAAGAAUAUUUGGUU 996 3405 CUAGAAAGAAUAUUUGGUU 996 3427AACCAAAUAUUCUUUCUAG 1219 3423 UUUCCUGUAUAGGAAUGAG 997 3423UUUCCUGUAUAGGAAUGAG 997 3445 CUCAUUCCUAUACAGGAAA 1220 3441GAUUAAUUCCUUUCCAGGU 998 3441 GAUUAAUUCCUUUCCAGGU 998 3463ACCUGGAAAGGAAUUAAUC 1221 3459 UAUUUUAUAAUUCUGGGAA 999 3459UAUUUUAUAAUUCUGGGAA 999 3481 UUCCCAGAAUUAUAAAAUA 1222 3477AGCAAAACCCAUGCCUCCC 1000 3477 AGCAAAACCCAUGCCUCCC 1000 3499GGGAGGCAUGGGUUUUGCU 1223 3495 CCCUAGCCAUUUUUACUGU 1001 3495CCCUAGCCAUUUUUACUGU 1001 3517 ACAGUAAAAAUGGCUAGGG 1224 3513UUAUCCUAUUUAGAUGGCC 1002 3513 UUAUCCUAUUUAGAUGGCC 1002 3535GGCCAUCUAAAUAGGAUAA 1225 3531 CAUGAAGAGGAUGCUGUGA 1003 3531CAUGAAGAGGAUGCUGUGA 1003 3553 UCACAGCAUCCUCUUCAUG 1226 3549AAAUUCCCAACAAACAUUG 1004 3549 AAAUUCCCAACAAACAUUG 1004 3571CAAUGUUUGUUGGGAAUUU 1227 3567 GAUGCUGACAGUCAUGCAG 1005 3567GAUGCUGACAGUCAUGCAG 1005 3589 CUGCAUGACUGUCAGCAUC 1228 3585GUCUGGGAGUGGGGAAGUG 1006 3585 GUCUGGGAGUGGGGAAGUG 1006 3607CACUUCCCCACUCCCAGAC 1229 3603 GAUCUUUUGUUCCCAUCCU 1007 3603GAUCUUUUGUUCCCAUCCU 1007 3625 AGGAUGGGAACAAAAGAUC 1230 3621UCUUCUUUUAGCAGUAAAA 1008 3621 UCUUCUUUUAGCAGUAAAA 1008 3643UUUUACUGCUAAAAGAAGA 1231 3639 AUAGCUGAGGGAAAAGGGA 1009 3639AUAGCUGAGGGAAAAGGGA 1009 3661 UCCCUUUUCCCUCAGCUAU 1232 3657AGGGAAAAGGAAGUUAUGG 1010 3657 AGGGAAAAGGAAGUUAUGG 1010 3679CCAUAACUUCCUUUUCCCU 1233 3675 GGAAUACCUGUGGUGGUUG 1011 3675GGAAUACCUGUGGUGGUUG 1011 3697 CAACCACCACAGGUAUUCC 1234 3693GUGAUCCCUAGGUCUUGGG 1012 3693 GUGAUCCCUAGGUCUUGGG 1012 3715CCCAAGACCUAGGGAUCAC 1235 3711 GAGCUCUUGGAGGUGUCUG 1013 3711GAGCUCUUGGAGGUGUCUG 1013 3733 CAGACACCUCCAAGAGCUC 1236 3729GUAUCAGUGGAUUUCCCAU 1014 3729 GUAUCAGUGGAUUUCCCAU 1014 3751AUGGGAAAUCCACUGAUAC 1237 3747 UCCCCUGUGGGAAAUUAGU 1015 3747UCCCCUGUGGGAAAUUAGU 1015 3769 ACUAAUUUCCCACAGGGGA 1238 3765UAGGCUCAUUUACUGUUUU 1016 3765 UAGGCUCAUUUACUGUUUU 1016 3787AAAACAGUAAAUGAGCCUA 1239 3783 UAGGUCUAGCCUAUGUGGA 1017 3783UAGGUCUAGCCUAUGUGGA 1017 3805 UCCACAUAGGCUAGACCUA 1240 3801AUUUUUUCCUAACAUACCU 1018 3801 AUUUUUUCCUAACAUACCU 1018 3823AGGUAUGUUAGGAAAAAAU 1241 3819 UAAGCAAACCCAGUGUCAG 1019 3819UAAGCAAACCCAGUGUCAG 1019 3841 CUGACACUGGGUUUGCUUA 1242 3837GGAUGGUAAUUCUUAUUCU 1020 3837 GGAUGGUAAUUCUUAUUCU 1020 3859AGAAUAAGAAUUACCAUCC 1243 3855 UUUCGUUCAGUUAAGUUUU 1021 3855UUUCGUUCAGUUAAGUUUU 1021 3877 AAAACUUAACUGAACGAAA 1244 3873UUCCCUUCAUCUGGGCACU 1022 3873 UUCCCUUCAUCUGGGCACU 1022 3895AGUGCCCAGAUGAAGGGAA 1245 3891 UGAAGGGAUAUGUGAAACA 1023 3891UGAAGGGAUAUGUGAAACA 1023 3913 UGUUUCACAUAUCCCUUCA 1246 3909AAUGUUAACAUUUUUGGUA 1024 3909 AAUGUUAACAUUUUUGGUA 1024 3931UACCAAAAAUGUUAACAUU 1247 3927 AGUCUUCAACCAGGGAUUG 1025 3927AGUCUUCAACCAGGGAUUG 1025 3949 CAAUCCCUGGUUGAAGACU 1248 3945GUUUCUGUUUAACUUCUUA 1026 3945 GUUUCUGUUUAACUUCUUA 1026 3967UAAGAAGUUAAACAGAAAC 1249 3963 AUAGGAAAGCUUGAGUAAA 1027 3963AUAGGAAAGCUUGAGUAAA 1027 3985 UUUACUCAAGCUUUCCUAU 1250 3981AAUAAAUAUUGUCUUUUUG 1028 3981 AAUAAAUAUUGUCUUUUUG 1028 4003CAAAAAGACAAUAUUUAUU 1251 3986 AUAUUGUCUUUUUGUAUGU 1029 3986AUAUUGUCUUUUUGUAUGU 1029 4008 ACAUACAAAAAGACAAUAU 1252 The 3′-ends ofthe Upper sequence and the Lower sequence of the siNA construct caninclude an overhang sequence, for example about 1, 2, 3, or 4nucleotides in length, preferably 2 nucleotides in length, wherein theoverhanging sequence of the lower sequence is optionally complementaryto a portion of the target sequence. The upper sequence is also referredto as the sense strand, whereas the lower sequence is also referred toas the antisense strand. The upper and lower sequences in the Table canfurther comprise a chemical modification having Formulae I-VII or anycombination thereof.

TABLE III Interleukin and Interleukin receptor Synthetic Modified siNAconstructs Target Seq Seq Pos Target ID Cmpd# Aliases Sequence ID IL2RG118 ACACCACAGCUGAUUUCUUCCUG 1253 IL2RG:120U21 sense siNAACCACAGCUGAUUUCUUCCTT 1311 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:132U21sense siNA UUCUUCCUGACCACUAUGCTT 1312 138 CUGACCACUAUGCCCACUGACUC 1255IL2RG:140U21 sense siNA GACCACUAUGCCCACUGACTT 1313 155UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:157U21 sense siNAACUCCCUCAGUGUUUCCACTT 1314 262 CCAACCUCACUCUGCAUUAUUGG 1257 IL2RG:264U21sense siNA AACCUCACUCUGCAUUAUUTT 1315 302 UGAUAAAGUCCAGAAGUGCAGCC 1258IL2RG:304U21 sense siNA AUAAAGUCCAGAAGUGCAGTT 1316 303GAUAAAGUCCAGAAGUGCAGCCA 1259 IL2RG:305U21 sense siNAUAAAGUCCAGAAGUGCAGCTT 1317 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:346U21sense siNA UCACUUCUGGCUGUCAGUUTT 1318 118 ACACCACAGCUGAUUUCUUCCUG 1253IL2RG:138L21 antisense siNA GGAAGAAAUCAGCUGUGGUTT 1319 (120C) 130AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNAGCAUAGUGGUCAGGAAGAATT 1320 (132C) 138 CUGACCACUAUGCCCACUGACUC 1255IL2RG:158L21 antisense siNA GUCAGUGGGCAUAGUGGUCTT 1321 (140C) 155UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNAGUGGAAACACUGAGGGAGUTT 1322 (157C) 262 CCAACCUCACUCUGCAUUAUUGG 1257IL2RG:282L21 antisense siNA AAUAAUGCAGAGUGAGGUUTT 1323 (264C) 302UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNACUGCACUUCUGGACUUUAUTT 1324 (304C) 303 GAUAAAGUCCAGAAGUGCAGCCA 1259IL2RG:323L21 antisense siNA GCUGCACUUCUGGACUUUATT 1325 (305C) 344AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNAAACUGACAGCCAGAAGUGATT 1326 (346C) 118 ACACCACAGCUGAUUUCUUCCUG 1253IL2RG:120U21 sense siNA B AccAcAGcuGAuuucuuccTT B 1327 stab04 130AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:132U21 sense siNA BuucuuccuGAccAcuAuGcTT B 1328 stab04 138 CUGACCACUAUGCCCACUGACUC 1255IL2RG:140U21 sense siNA B GAccAcuAuGcccAcuGAcTT B 1329 stab04 155UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:157U21 sense siNA BAcucccucAGuGuuuccAcTT B 1330 stab04 262 CCAACCUCACUCUGCAUUAUUGG 1257IL2RG:264U21 sense siNA B AAccucAcucuGcAuuAuuTT B 1331 stab04 302UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:304U21 sense siNA BAuAAAGuccAGAAGuGcAGTT B 1332 stab04 303 GAUAAAGUCCAGAAGUGCAGCCA 1259IL2RG:305U21 sense siNA B uAAAGuccAGAAGuGcAGcTT B 1333 stab04 344AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:346U21 sense siNA BucAcuucuGGcuGucAGuuTT B 1334 stab04 118 ACACCACAGCUGAUUUCUUCCUG 1253IL2RG:138L21 antisense siNA GGAAGAAAucAGcuGuGGuTsT 1335 (120C) stab05130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNAGcAuAGuGGucAGGAAGAATsT 1336 (132C) stab05 138 CUGACCACUAUGCCCACUGACUC1255 IL2RG:158L21 antisense siNA GucAGuGGGcAuAGuGGucTsT 1337 (140C)stab05 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNAGuGGAAAcAcuGAGGGAGuTsT 1338 (157C) stab05 262 CCAACCUCACUCUGCAUUAUUGG1257 IL2RG:282L21 antisense siNA AAuAAuGcAGAGuGAGGuuTsT 1339 (264C)stab05 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNAcuGcAcuucuGGAcuuuAuTsT 1340 (304C) stab05 303 GAUAAAGUCCAGAAGUGCAGCCA1259 IL2RG:323L21 antisense siNA GcuGcAcuucuGGAcuuuATsT 1341 (305C)stab05 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNAAAcuGAcAGccAGAAGuGATsT 1342 (346C) stab05 118 ACACCACAGCUGAUUUCUUCCUG1253 IL2RG:120U21 sense siNA B AccAcAGcuGAuuucuuccTT B 1343 stab07 130AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:132U21 sense siNA BuucuuccuGAccAcuAuGcTT B 1344 stab07 138 CUGACCACUAUGCCCACUGACUC 1255IL2RG:140U21 sense siNA B GAccAcuAuGcccAcuGAcTT B 1345 stab07 155UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:157U21 sense siNA BAcucccucAGuGuuuccAcTT B 1346 stab07 262 CCAACCUCACUCUGCAUUAUUGG 1257IL2RG:264U21 sense siNA B AAccucAcucuGcAuuAuuTT B 1347 stab07 302UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:304U21 sense siNA BAuAAAGuccAGAAGuGcAGTT B 1348 stab07 303 GAUAAAGUCCAGAAGUGCAGCCA 1259IL2RG:305U21 sense siNA B uAAAGuccAGAAGuGcAGcTT B 1349 stab07 344AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:346U21 sense siNA BucAcuucuGGcuGucAGuuTT B 1350 stab07 118 ACACCACAGCUGAUUUCUUCCUG 1253IL2RG:138L21 antisense siNA GGAAGAAAucAGcuGuGGuTsT 1351 (120C) stab11130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNAGcAuAGuGGucAGGAAGAATsT 1352 (132C) stab11 138 CUGACCACUAUGCCCACUGACUC1255 IL2RG:158L21 antisense siNA GucAGuGGGcAuAGuGGucTsT 1353 (140C)stab11 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNAGuGGAAAcAcuGAGGGAGuTsT 1354 (157C) stab11 262 CCAACCUCACUCUGCAUUAUUGG1257 IL2RG:282L21 antisense siNA AAuAAuGcAGAGuGAGGuuTsT 1355 (264C)stab11 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNAcuGcAcuucuGGAcuuuAuTsT 1356 (304C) stab11 303 GAUAAAGUCCAGAAGUGCAGCCA1259 IL2RG:323L21 antisense siNA GcuGcAcuucuGGAcuuuATsT 1357 (305C)stab11 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNAAAcuGAcAGccAGAAGuGATsT 1358 (346C) stab11 118 ACACCACAGCUGAUUUCUUCCUG1253 IL2RG:120U21 sense siNA B AccAcAGcuGAuuucuuccTT B 1359 stab18 130AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:132U21 sense siNA BuucuuccuGAccAcuAuGcTT B 1360 stab18 138 CUGACCACUAUGCCCACUGACUC 1255IL2RG:140U21 sense siNA B GAccAcuAuGcccAcuGAcTT B 1361 stab18 155UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:157U21 sense siNA BAcucccucAGuGuuuccAcTT B 1362 stab18 262 CCAACCUCACUCUGCAUUAUUGG 1257IL2RG:264U21 sense siNA B AAccucAcucuGcAuuAuuTT B 1363 stab18 302UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:304U21 sense siNA BAuAAAGuccAGAAGUuGcAGTT B 1364 stab18 303 GAUAAAGUCCAGAAGUGCAGCCA 1259IL2RG:305U21 sense siNA B uAAAGuccAGAAGuGcAGcTT B 1365 stab18 344AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:346U21 sense siNA BucAcuucuGGcuGucAGuuTT B 1366 stab18 118 ACACCACAGCUGAUUUCUUCCUG 1253IL2RG:138L21 antisense siNA GGAAGAAAucAGcuGuGGuTsT 1367 (120C) stab08130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNAGcAuAGuGGucAGGAAGAATsT 1368 (132C) stab08 138 CUGACCACUAUGCCCACUGACUC1255 IL2RG:158L21 antisense siNA GucAGuGGGcAuAGuGGucTsT 1369 (140C)stab08 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNAGuGGAAAcAcuGAGGGAGuTsT 1370 (157C) stab08 262 CCAACCUCACUCUGCAUUAUUGG1257 IL2RG:282L21 antisense siNA AAuAAuGcAGAGuGAGGuuTsT 1371 (264C)stab08 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNAcuGcAcuucuGGAcuuuAuTsT 1372 (304C) stab08 303 GAUAAAGUCCAGAAGUGCAGCCA1259 IL2RG:323L21 antisense siNA GcuGcAcuucuGGAcuuuATsT 1373 (305C)stab08 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNAAAcuGAcAGccAGAAGuGATsT 1374 (346C) stab08 118 ACACCACAGCUGAUUUCUUCCUG1253 IL2RG:120U21 sense siNA B ACCACAGCUGAUUUCUUCCTT B 1375 stab09 130AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:132U21 sense siNA BUUCUUCCUGACCACUAUGCTT B 1376 stab09 138 CUGACCACUAUGCCCACUGACUC 1255IL2RG:140U21 sense siNA B GACCACUAUGCCCACUGACTT B 1377 stab09 155UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:157U21 sense siNA BACUCCCUCAGUGUUUCCACTT B 1378 stab09 262 CCAACCUCACUCUGCAUUAUUGG 1257IL2RG:264U21 sense siNA B AACCUCACUCUGCAUUAUUTT B 1379 stab09 302UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:304U21 sense siNA BAUAAAGUCCAGAAGUGCAGTT B 1380 stab09 303 GAUAAAGUCCAGAAGUGCAGCCA 1259IL2RG:305U21 sense siNA B UAAAGUCCAGAAGUGCAGCTT B 1381 stab09 344AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:346U21 sense siNA BUCACUUCUGGCUGUCAGUUTT B 1382 stab09 118 ACACCACAGCUGAUUUCUUCCUG 1253IL2RG:138L21 antisense siNA GGAAGAAAUCAGCUGUGGUTsT 1383 (120C) stab10130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNAGCAUAGUGGUCAGGAAGAATsT 1384 (132C) stab10 138 CUGACCACUAUGCCCACUGACUC1255 IL2RG:158L21 antisense siNA GUCAGUGGGCAUAGUGGUCTsT 1385 (140C)stab10 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNAGUGGAAACACUGAGGGAGUTsT 1386 (157C) stab10 262 CCAACCUCACUCUGCAUUAUUGG1257 IL2RG:282L21 antisense siNA AAUAAUGCAGAGUGAGGUUTsT 1387 (264C)stab10 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNACUGCACUUCUGGACUUUAUTsT 1388 (304C) stab10 303 GAUAAAGUCCAGAAGUGCAGCCA1259 IL2RG:323L21 antisense siNA GCUGCACUUCUGGACUUUATsT 1389 (305C)stab10 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNAAACUGACAGCCAGAAGUGATsT 1390 (346C) stab10 118 ACACCACAGCUGAUUUCUUCCUG1253 IL2RG:138L21 antisense siNA GGAAGAAAucAGcuGuGGuTT B 1391 (120C)stab19 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNAGcAuAGuGGucAGGAAGAATT B 1392 (132C) stab19 138 CUGACCACUAUGCCCACUGACUC1255 IL2RG:158L21 antisense siNA GucAGuGGGcAuAGuGGucTT B 1393 (140C)stab19 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNAGuGGAAAcAcuGAGGGAGuTT B 1394 (157C) stab19 262 CCAACCUCACUCUGCAUUAUUGG1257 IL2RG:282L21 antisense siNA AAuAAuGcAGAGuGAGGuuTT B 1395 (264C)stab19 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNAcuGcAcuucuGGAcuuuAuTT B 1396 (304C) stab19 303 GAUAAAGUCCAGAAGUGCAGCCA1259 IL2RG:323L21 antisense siNA GcuGcAcuucuGGAcuuuATT B 1397 (305C)stab19 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNAAAcuGAcAGccAGAAGuGATT B 1398 (346C) stab19 118 ACACCACAGCUGAUUUCUUCCUG1253 IL2RG:138L21 antisense siNA GGAAGAAAUCAGCUGUGGUTT B 1399 (120C)stab22 130 AUUUCUUCCUGACCACUAUGCCC 1254 IL2RG:150L21 antisense siNAGCAUAGUGGUCAGGAAGAATT B 1400 (132C) stab22 138 CUGACCACUAUGCCCACUGACUC1255 IL2RG:158L21 antisense siNA GUCAGUGGGCAUAGUGGUCTT B 1401 (140C)stab22 155 UGACUCCCUCAGUGUUUCCACUC 1256 IL2RG:175L21 antisense siNAGUGGAAACACUGAGGGAGUTT B 1402 (157C) stab22 262 CCAACCUCACUCUGCAUUAUUGG1257 IL2RG:282L21 antisense siNA AAUAAUGCAGAGUGAGGUUTT B 1403 (264C)stab22 302 UGAUAAAGUCCAGAAGUGCAGCC 1258 IL2RG:322L21 antisense siNACUGCACUUCUGGACUUUAUTT B 1404 (304C) stab22 303 GAUAAAGUCCAGAAGUGCAGCCA1259 IL2RG:323L21 antisense siNA GCUGCACUUCUGGACUUUATT B 1405 (305C)stab22 344 AAUCACUUCUGGCUGUCAGUUGC 1260 IL2RG:364L21 antisense siNAAACUGACAGCCAGAAGUGATT B 1406 (346C) stab22 IL4 487CAGCCUCACAGAGCAGAAGACUC 1269 IL4:489U21 sense siNA GCCUCACAGAGCAGAAGACTT1407 489 GCCUCACAGAGCAGAAGACUCUG 1270 IL4:491U21 sense siNACUCACAGAGCAGAAGACUCTT 1408 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:518U21sense siNA GAGUUGACCGUAACAGACATT 1409 526 CGUAACAGACAUCUUUGCUGCCU 1272IL4:528U21 sense siNA UAACAGACAUCUUUGCUGCTT 1410 545GCCUCCAAGAACACAACUGAGAA 1273 IL4:547U21 sense siNA CUCCAAGAACACAACUGAGTT1411 606 UCUACAGCCACCAUGAGAAGGAC 1274 IL4:608U21 sense siNAUACAGCCACCAUGAGAAGGTT 1412 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:730U21sense siNA GAAUUCCUGUCCUGUGAAGTT 1413 745 GAAGGAAGCCAACCAGAGUACGU 1276IL4:747U21 sense siNA AGGAAGCCAACCAGAGUACTT 1414 487CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNAGUCUUCUGCUCUGUGAGGCTT 1415 (489C) 489 GCCUCACAGAGCAGAAGACUCUG 1270IL4:509L21 antisense siNA GAGUCUUCUGCUCUGUGAGTT 1416 (491C) 516CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNAUGUCUGUUACGGUCAACUCTT 1417 (518C) 526 CGUAACAGACAUCUUUGCUGCCU 1272IL4:546L21 antisense siNA GCAGCAAAGAUGUCUGUUATT 1418 (528C) 545GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNACUCAGUUGUGUUCUUGGAGTT 1419 (547C) 606 UCUACAGCCACCAUGAGAAGGAC 1274IL4:626L21 antisense siNA CCUUCUCAUGGUGGCUGUATT 1420 (608C) 728UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNACUUCACAGGACAGGAAUUCTT 1421 (730C) 745 GAAGGAAGCCAACCAGAGUACGU 1276IL4:765L21 antisense siNA GUACUCUGGUUGGCUUCCUTT 1422 (747C) 487CAGCCUCACAGAGCAGAAGACUC 1269 IL4:489U21 sense siNA BGccucAcAGAGcAGAAGAcTT B 1423 stab04 489 GCCUCACAGAGCAGAAGACUCUG 1270IL4:491U21 sense siNA B cucAcAGAGcAGAAGAcucTT B 1424 stab04 516CCGAGUUGACCGUAACAGACAUC 1271 IL4:518U21 sense siNA BGAGuuGAccGuAAcAGAcATT B 1425 stab04 526 CGUAACAGACAUCUUUGCUGCCU 1272IL4:528U21 sense siNA B uAAcAGAcAucuuuGcuGcTT B 1426 stab04 545GCCUCCAAGAACACAACUGAGAA 1273 IL4:547U21 sense siNA BcuccAAGAAcAcAAcuGAGTT B 1427 stab04 606 UCUACAGCCACCAUGAGAAGGAC 1274IL4:608U21 sense siNA B uAcAGccAccAuGAGAAGGTT B 1428 stab04 728UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:730U21 sense siNA BGAAuuccuGuccuGuGAAGTT B 1429 stab04 745 GAAGGAAGCCAACCAGAGUACGU 1276IL4:747U21 sense siNA B AGGAAGccAAccAGAGuAcTT B 1430 stab04 487CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNAGucuucuGcucuGuGAGGcTsT 1431 (489C) stab05 489 GCCUCACAGAGCAGAAGACUCUG1270 IL4:509L21 antisense siNA GAGucuucuGcucuGuGAGTsT 1432 (491C) stab05516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNAuGucuGuuAcGGucAAcucTsT 1433 (518C) stab05 526 CGUAACAGACAUCUUUGCUGCCU1272 IL4:546L21 antisense siNA GcAGcAAAGAuGucuGuuATsT 1434 (528C) stab05545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNAcucAGuuGuGuucuuGGAGTsT 1435 (547C) stab05 606 UCUACAGCCACCAUGAGAAGGAC1274 IL4:626L21 antisense siNA ccuucucAuGGuGGcuGuATsT 1436 (608C) stab05728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNAcuucAcAGGAcAGGAAuucTsT 1437 (730C) stab05 745 GAAGGAAGCCAACCAGAGUACGU1276 IL4:765L21 antisense siNA GuAcucuGGuuGGcuuccuTsT 1438 (747C) stab05487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:489U21 sense siNA BGccucAcAGAGcAGAAGAcTT B 1439 stab07 489 GCCUCACAGAGCAGAAGACUCUG 1270IL4:491U21 sense siNA B cucAcAGAGcAGAAGAcucTT B 1440 stab07 516CCGAGUUGACCGUAACAGACAUC 1271 IL4:518U21 sense siNA BGAGuuGAccGuAAcAGAcATT B 1441 stab07 526 CGUAACAGACAUCUUUGCUGCCU 1272IL4:528U21 sense siNA B uAAcAGAcAucuuuGcuGcTT B 1442 stab07 545GCCUCCAAGAACACAACUGAGAA 1273 IL4:547U21 sense siNA BcuccAAGAAcAcAAcuGAGTT B 1443 stab07 606 UCUACAGCCACCAUGAGAAGGAC 1274IL4:608U21 sense siNA B uAcAGccAccAuGAGAAGGTT B 1444 stab07 728UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:730U21 sense siNA BGAAuuccuGuccuGuGAAGTT B 1445 stab07 745 GAAGGAAGCCAACCAGAGUACGU 1276IL4:747U21 sense siNA B AGGAAGccAAccAGAGuAcTT B 1446 stab07 487CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNAGucuucuGcucuGuGAGGcTsT 1447 (489C) stab11 489 GCCUCACAGAGCAGAAGACUCUG1270 IL4:509L21 antisense siNA GAGucuucuGcucuGuGAGTsT 1448 (491C) stab11516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNAuGucuGuuAcGGucAAcucTsT 1449 (518C) stab11 526 CGUAACAGACAUCUUUGCUGCCU1272 IL4:546L21 antisense siNA GcAGcAAAGAuGucuGuuATsT 1450 (528C) stab11545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNAcucAGuuGuGuucuuGGAGTsT 1451 (547C) stab11 606 UCUACAGCCACCAUGAGAAGGAC1274 IL4:626L21 antisense siNA ccuucucAuGGuGGcuGuATsT 1452 (608C) stab11728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNAcuucAcAGGAcAGGAAuucTsT 1453 (730C) stab11 745 GAAGGAAGCCAACCAGAGUACGU1276 IL4:765L21 antisense siNA GuAcucuGGuuGGcuuccuTsT 1454 (747C)stab11487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:489U21 sense siNA BGccucAcAGAGcAGAAGAcTT B 1455 stab18 489 GCCUCACAGAGCAGAAGACUCUG 1270IL4:491U21 sense siNA B cucAcAGAGcAGAAGAcucTT B 1456 stab18 516CCGAGUUGACCGUAACAGACAUC 1271 IL4:518U21 sense siNA BGAGuuGAccGuAAcAGAcATT B 1457 stab18 526 CGUAACAGACAUCUUUGCUGCCU 1272IL4:528U21 sense siNA B uAAcAGAcAucuuuGcuGcTT B 1458 stab18 545GCCUCCAAGAACACAACUGAGAA 1273 IL4:547U21 sense siNA BcuccAAGAAcAcAAcuGAGTT B 1459 stab18 606 UCUACAGCCACCAUGAGAAGGAC 1274IL4:608U21 sense siNA B uAcAGccAccAuGAGAAGGTT B 1460 stab18 728UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:730U21 sense siNA BGAAuuccuGuccuGuGAAGTT B 1461 stab18 745 GAAGGAAGCCAACCAGAGUACGU 1276IL4:747U21 sense siNA B AGGAAGccAAccAGAGuAcTT B 1462 stab18 487CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNAGucuucuGcucuGuGAGGcTsT 1463 (489C) stab08 489 GCCUCACAGAGCAGAAGACUCUG1270 IL4:509L21 antisense siNA GAGucuucuGcucuGuGAGTsT 1464 (491C) stab08516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNAuGucuGuuAcGGucAAcucTsT 1465 (518C) stab08 526 CGUAACAGACAUCUUUGCUGCCU1272 IL4:546L21 antisense siNA GcAGcAAAGAuGucuGuuATsT 1466 (528C) stab08545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNAcucAGuuGuGuucuuGGAGTsT 1467 (547C) stab08 606 UCUACAGCCACCAUGAGAAGGAC1274 IL4:626L21 antisense siNA ccuucucAuGGuGGcuGuATsT 1468 (608C) stab08728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNAcuucAcAGGAcAGGAAuucTsT 1469 (730C) stabo8 745 GAAGGAAGCCAACCAGAGUACGU1276 IL4:765L21 antisense siNA GuAcucuGGuuGGcuuccuTsT 1470 (747C) stab08487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:489U21 sense siNA BGCCUCACAGAGCAGAAGACTT B 1471 stab09 489 GCCUCACAGAGCAGAAGACUCUG 1270IL4:491U21 sense siNA B CUCACAGAGCAGAAGACUCTT B 1472 stab09 516CCGAGUUGACCGUAACAGACAUC 1271 IL4:518U21 sense siNA BGAGUUGACCGUAACAGACATT B 1473 stab09 526 CGUAACAGACAUCUUUGCUGCCU 1272IL4:528U21 sense siNA B UAACAGACAUCUUUGCUGCTT B 1474 stab09 545GCCUCCAAGAACACAACUGAGAA 1273 IL4:547U21 sense siNA BCUCCAAGAACACAACUGAGTT B 1475 stab09 606 UCUACAGCCACCAUGAGAAGGAC 1274IL4:608U21 sense siNA B UACAGCCACCAUGAGAAGGTT B 1476 stab09 728UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:730U21 sense siNA BGAAUUCCUGUCCUGUGAAGTT B 1477 stab09 745 GAAGGAAGCCAACCAGAGUACGU 1276IL4:747U21 sense siNA B AGGAAGCCAACCAGAGUACTT B 1478 stab09 487CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNAGUCUUCUGCUCUGUGAGGCTsT 1479 (489C) stab10 489 GCCUCACAGAGCAGAAGACUCUG1270 IL4:509L21 antisense siNA GAGUCUUCUGCUCUGUGAGTsT 1480 (491C) stab10516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNAUGUCUGUUACGGUCAACUCTsT 1481 (518C) stab10 526 CGUAACAGACAUCUUUGCUGCCU1272 IL4:546L21 antisense siNA GCAGCAAAGAUGUCUGUUATsT 1482 (528C) stab10545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNACUCAGUUGUGUUCUUGGAGTsT 1483 (547C) stab10 606 UCUACAGCCACCAUGAGAAGGAC1274 IL4:626L21 antisense siNA CCUUCUCAUGGUGGCUGUATsT 1484 (608C) stab10728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNACUUCACAGGACAGGAAUUCTsT 1485 (730C) stab10 745 GAAGGAAGCCAACCAGAGUACGU1276 IL4:765L21 antisense siNA GUACUCUGGUUGGCUUCCUTsT 1486 (747C) stab10487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNAGucuucuGcucuGuGAGGcTT B 1487 (489C) stab19 489 GCCUCACAGAGCAGAAGACUCUG1270 IL4:509L21 antisense siNA GAGucuucuGcucuGuGAGTT B 1488 (491C)stab19 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNAuGucuGuuAcGGucAAcucTT B 1489 (518C) stab19 526 CGUAACAGACAUCUUUGCUGCCU1272 IL4:546L21 antisense siNA GcAGcAAAGAuGucuGuuATT B 1490 (528C)stab19 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNAcucAGuuGuGuucuuGGAGTT B 1491 (547C) stab19 606 UCUACAGCCACCAUGAGAAGGAC1274 IL4:626L21 antisense siNA ccuucucAuGGuGGcuGuATT B 1492 (6C8C)stab19 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNAcuucAcAGGAcAGGAAuucTT B 1493 (730C) stab19 745 GAAGGAAGCCAACCAGAGUACGU1276 IL4:765L21 antisense siNA GuAcucuGGuuGGcuuccuTT B 1494 (747C)stab19 487 CAGCCUCACAGAGCAGAAGACUC 1269 IL4:507L21 antisense siNAGUCUUCUGCUCUGUGAGGCTT B 1495 (489C) stab22 489 GCCUCACAGAGCAGAAGACUCUG1270 IL4:509L21 antisense siNA GAGUCUUCUGCUCUGUGAGTT B 1496 (491C)stab22 516 CCGAGUUGACCGUAACAGACAUC 1271 IL4:536L21 antisense siNAUGUCUGUUACGGUCAACUCTT B 1497 (518C) stab22 526 CGUAACAGACAUCUUUGCUGCCU1272 IL4:546L21 antisense siNA GCAGCAAAGAUGUCUGUUATT B 1498 (528C)stab22 545 GCCUCCAAGAACACAACUGAGAA 1273 IL4:565L21 antisense siNACUCAGUUGUGUUCUUGGAGTT B 1499 (547C) stab22 606 UCUACAGCCACCAUGAGAAGGAC1274 IL4:626L21 antisense siNA CCUUCUCAUGGUGGCUGUATT B 1500 (608C)stab22 728 UUGAAUUCCUGUCCUGUGAAGGA 1275 IL4:748L21 antisense siNACUUCACAGGACAGGAAUUCTT B 1501 (730C) stab22 745 GAAGGAAGCCAACCAGAGUACGU1276 IL4:765L21 antisense siNA GUACUCUGGUUGGCUUCCUTT B 1502 (747C)stab22 IL4R 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:471U21 sense siNAAUACACUGGACCUGUGGGCTT 1503 551 CCAGGAAACCUGACAGUUCACAC 1278 IL4R:553U21sense siNA AGGAAACCUGACAGUUCACTT 1504 1119 AGCACAACAUGAAAAGGGAUGAA 1279IL4R:1121U21 sense siNA CACAACAUGAAAAGGGAUGTT 1505 1120GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1122U21 sense siNAACAACAUGAAAAGGGAUGATT 1506 1132 AAGGGAUGAAGAUCCUCACAAGG 1281IL4R:1134U21 sense siNA GGGAUGAAGAUCCUCACAATT 1507 3130UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3132U21 sense siNAGGGAAAUCGAUGAGAAAUUTT 1508 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283IL4R:3133U21 sense siNA GGAAAUCGAUGAGAAAUUGTT 1509 3169UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3171U21 sense siNAAUUGCCUAGAGGUGCUCAUTT 1510 469 CUAUACACUGGACCUGUGGGCUG 1277 IL4R:489L21antisense siNA GCCCACAGGUCCAGUGUAUTT 1511 (471C) 551CCAGGAAACCUGACAGUUCACAC 1278 IL4R:57IL21 antisense siNAGUGAACUGUCAGGUUUCCUTT 1512 (553C) 1119 AGCACAACAUGAAAAGGGAUGAA 1279IL4R:1139L21 antisense siNA CAUCCCUUUUCAUGUUGUGTT 1513 (1121C) 1120GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1140L21 antisense siNAUCAUCCCUUUUCAUGUUGUTT 1514 (1122C) 1132 AAGGGAUGAAGAUCCUCACAAGG 1281IL4R:1152L21 antisense siNA UUGUGAGGAUCUUCAUCCCTT 1515 (1134C) 3130UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3150L21 antisense siNAAAUUUCUCAUCGAUUUCCCTT 1516 (3132C) 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283IL4R:315IL21 antisense siNA CAAUUUCUCAUCGAUUUCCTT 1517 (3133C) 3169UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3189L21 antisense siNAAUGAGCACCUCUAGGCAAUTT 1518 (3171C) 469 CUAUACACUGGACCUGUGGGCUG 1277IL4R:471U21 sense siNA B AuAcAcuGGAccuGuGGGcTT B 1519 stab04 551CCAGGAAACCUGACAGUUCACAC 1278 IL4R:553U21 sense siNA BAGGAAAccuGAcAGuucAcTT B 1520 stab04 1119 AGCACAACAUGAAAAGGGAUGAA 1279IL4R:1121U21 sense siNA B cAcAAcAuGAAAAGGGAuGTT B 1521 stab04 1120GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1122U21 sense siNA BAcAAcAuGAAAAGGGAuGATT B 1522 stab04 1132 AAGGGAUGAAGAUCCUCACAAGG 1281IL4R:1134U21 sense siNA B GGGAuGAAGAuccucAcAATT B 1523 stab04 3130UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3132U21 sense siNA BGGGAAAucGAuGAGAAAuuTT B 1524 stab04 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283IL4R:3133U21 sense siNA B GGAAAucGAuGAGAAAuuGTT B 1525 stab04 3169UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3171U21 sense siNA BAuuGccuAGAGGuGcucAuTT B 1526 stab04 469 CUAUACACUGGACCUGUGGGCUG 1277IL4R:489L21 antisense siNA GcccAcAGGuccAGuGuAuTsT 1527 (471C) stab05 551CCAGGAAACCUGACAGUUCACAC 1278 IL4R:571L21 antisense siNAGuGAAcuGucAGGuuuccuTsT 1528 (553C) stab05 1119 AGCACAACAUGAAAAGGGAUGAA1279 IL4R:1139L21 antisense siNA cAucccuuuucAuGuuGuGTsT 1529 (1121C)stab05 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1140L21 antisense siNAucAucccuuuucAuGuuGuTsT 1530 (1122C) stab05 1132 AAGGGAUGAAGAUCCUCACAAGG1281 IL4R:1152L21 antisense siNA uuGuGAGGAucuucAucccTsT 1531 (1134C)stab05 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3150L21 antisense siNAAAuuucucAucGAuuucccTsT 1532 (3132C) stab05 3131 UGGGAAAUCGAUGAGAAAUUGAA1283 IL4R:315IL21 antisense siNA cAAuuucucAucGAuuuccTsT 1533 (3133C)stab05 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3189L21 antisense siNAAuGAGcAccucuAGGcAAuTsT 1534 (3171C) stab05 469 CUAUACACUGGACCUGUGGGCUG1277 IL4R:471U21 sense siNA B AuAcAcuGGAccuGuGGGcTT B 1535 stab07 551CCAGGAAACCUGACAGUUCACAC 1278 IL4R:553U21 sense siNA BAGGAAAccuGAcAGuucAcTT B 1536 stab07 1119 AGCACAACAUGAAAAGGGAUGAA 1279IL4R:1121U21 sense siNA B cAcAAcAuGAAAAGGGAuGTT B 1537 stab07 1120GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1122U21 sense siNA BAcAAcAuGAAAAGGGAuGATT B 1538 stab07 1132 AAGGGAUGAAGAUCCUCACAAGG 1281IL4R:1134U21 sense siNA B GGGAuGAAGAuccucAcAATT B 1539 stab07 3130UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3132U21 sense siNA BGGGAAAucGAuGAGAAAuuTT B 1540 stab07 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283IL4R:3133U21 sense siNA B GGAAAucGAuGAGAAAuuGTT B 1541 stab07 3169UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3171U21 sense siNA BAuuGccuAGAGGuGcucAuTT B 1542 stab07 469 CUAUACACUGGACCUGUGGGCUG 1277IL4R:489L21 antisense siNA GcccAcAGGuccAGuGuAuTsT 1543 (471C) stab11 551CCAGGAAACCUGACAGUUCACAC 1278 IL4R:571L21 antisense siNAGuGAAcuGucAGGuuuccuTsT 1544 (553C) stab11 1119 AGCACAACAUGAAAAGGGAUGAA1279 IL4R:1139L21 antisense siNA cAucccuuuucAuGuuGuGTsT 1545 (1121C)stab11 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1140L21 antisense siNAucAucccuuuucAuGuuGuTsT 1546 (1122C) stab11 1132 AAGGGAUGAAGAUCCUCACAAGG1281 IL4R:1152L21 antisense siNA uuGuGAGGAucuucAucccTsT 1547 (1134C)stab11 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3150L21 antisense siNAAAuuucucAucGAuuucccTsT 1548 (3132C) stab11 3131 UGGGAAAUCGAUGAGAAAUUGAA1283 IL4R:315IL21 antisense siNA cAAuuucucAucGAuuuccTsT 1549 (3133C)stab11 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3189L21 antisense siNAAuGAGcAccucuAGGcAAuTsT 1550 (3171C) stab11 469 CUAUACACUGGACCUGUGGGCUG1277 IL4R:471U21 sense siNA B AuAcAcuGGAccuGuGGGcTTB 1551 stab18 551CCAGGAAACCUGACAGUUCACAC 1278 IL4R:553U21 sense siNA BAGGAAAccuGAcAGuucAcTT B 1552 stab18 1119 AGCACAACAUGAAAAGGGAUGAA 1279IL4R:1121U21 sense siNA B cAcAAcAuGAAAAGGGAuGTT B 1553 stab18 1120GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1122U21 sense siNA BAcAAcAuGAAAAGGGAuGATT B 1554 stab18 1132 AAGGGAUGAAGAUCCUCACAAGG 1281IL4R:1134U21 sense siNA B GGGAuGAAGAuccucAcAATT B 1555 stab18 3130UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3132U21 sense siNA BGGGAAAucGAuGAGAAAuuTT B 1556 stab18 3131 UGGGAAAUCGAUGAGAAAUUGAA 1283IL4R:3133U21 sense siNA B GGAAAucGAuGAGAAAuuGTT B 1557 stab18 3169UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3171U21 sense siNA BAuuGccuAGAGGuGcucAuTT B 1558 stab18 469 CUAUACACUGGACCUGUGGGCUG 1277IL4R:489L21 antisense siNA GcccAcAGGuccAGuGuAuTsT 1559 (471C) stab08 551CCAGGAAACCUGACAGUUCACAC 1278 IL4R:571L21 antisense siNAGuGAAcuGucAGGuuuccuTsT 1560 (553C) stab08 1119 AGCACAACAUGAAAAGGGAUGAA1279 IL4R:1139L21 antisense siNA cAucccuuuucAuGuuGuGTsT 1561 (1121C)stab08 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1140L21 antisense siNAucAucccuuuucAuGuuGuTsT 1562 (1122C) stab08 1132 AAGGGAUGAAGAUCCUCACAAGG1281 IL4R:1152L21 antisense siNA uuGuGAGGAucuucAucccTsT 1563 (1134C)stab08 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3150L21 antisense siNAAAuuucucAucGAuuucccTsT 1564 (3132C) stab08 3131 UGGGAAAUCGAUGAGAAAUUGAA1283 IL4R:315IL21 antisense siNA cAAuuucucAucGAuuuccTsT 1565 (3133C)stab08 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3189L21 antisense siNAAuGAGcAccucuAGGcAAuTsT 1566 (3171C) stab08 469 CUAUACACUGGACCUGUGGGCUG1277 36729 IL4R:471U21 sense siNA B AUACACUGGACCUGUGGGCTT B 1567 stab09551 CCAGGAAACCUGACAGUUCACAC 1278 36730 IL4R:553U21 sense siNA BAGGAAACCUGACAGUUCACTT B 1568 stab09 1119 AGCACAACAUGAAAAGGGAUGAA 127936731 IL4R:1121U21 sense siNA B CACAACAUGAAAAGGGAUGTT B 1569 stab09 1120GCACAACAUGAAAAGGGAUGAAG 1280 36732 IL4R:1122U21 sense siNA BACAACAUGAAAAGGGAUGATT B 1570 stab09 1132 AAGGGAUGAAGAUCCUCACAAGG 128136733 IL4R:1134U21 sense siNA B GGGAUGAAGAUCCUCACAATT B 1571 stab09 3130UUGGGAAAUCGAUGAGAAAUUGA 1282 36734 IL4R:3132U21 sense siNA BGGGAAAUCGAUGAGAAAUUTT B 1572 stab09 3131 UGGGAAAUCGAUGAGAAAUUGAA 128336735 IL4R:3133U21 sense siNA B GGAAAUCGAUGAGAAAUUGTT B 1573 stab09 3169UCAUUGCCUAGAGGUGCUCAUUC 1284 36736 IL4R:3171U21 sense siNA BAUUGCCUAGAGGUGCUCAUTT B 1574 stab09 469 CUAUACACUGGACCUGUGGGCUG 1277IL4R:489L21 antisense siNA GCCCACAGGUCCAGUGUAUTsT 1575 (471C) stab10 551CCAGGAAACCUGACAGUUCACAC 1278 IL4R:571L21 antisense siNAGUGAACUGUCAGGUUUCCUTsT 1576 (553C) stab10 1119 AGCACAACAUGAAAAGGGAUGAA1279 IL4R:1139L21 antisense siNA CAUCCCUUUUCAUGUUGUGTsT 1577 (1121C)stab10 1120 GCACAACAUGAAAAGGGAUGAAG 1280 IL4R:1140L21 antisense siNAUCAUCCCUUUUCAUGUUGUTsT 1578 (1122C) stab10 1132 AAGGGAUGAAGAUCCUCACAAGG1281 IL4R:1152L21 antisense siNA UUGUGAGGAUCUUCAUCCCTsT 1579 (1134C)stab10 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 IL4R:3150L21 antisense siNAAAUUUCUCAUCGAUUUCCCTsT 1580 (3132C) stab10 3131 UGGGAAAUCGAUGAGAAAUUGAA1283 IL4R:315IL21 antisense siNA CAAUUUCUCAUCGAUUUCCTsT 1581 (3133C)stab10 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 IL4R:3189L21 antisense siNAAUGAGCACCUCUAGGCAAUTsT 1582 (3171C) stab10 469 CUAUACACUGGACCUGUGGGCUG1277 36737 IL4R:489L21 antisense siNA GcccAcAGGuccAGuGuAuTT B 1583(471C) stab19 551 CCAGGAAACCUGACAGUUCACAC 1278 36738 IL4R:571L21antisense siNA GuGAAcuGucAGGuuuccuTT B 1584 (553C) stab19 1119AGCACAACAUGAAAAGGGAUGAA 1279 36739 IL4R:1139L21 antisense siNAcAucccuuuucAuGuuGuGTT B 1585 (1121C) stab19 1120 GCACAACAUGAAAAGGGAUGAAG1280 36740 IL4R:1140L21 antisense siNA ucAucccuuuucAuGuuGuTT B 1586(1122C) stab19 1132 AAGGGAUGAAGAUCCUCACAAGG 1281 36741 IL4R:1152L21antisense siNA uuGuGAGGAucuucAucccTT B 1587 (1134C) stab19 3130UUGGGAAAUCGAUGAGAAAUUGA 1282 36742 IL4R:3150L21 antisense siNAAAuuucucAucGAuuucccTT B 1588 (3132C) stab19 3131 UGGGAAAUCGAUGAGAAAUUGAA1283 36743 IL4R:315IL21 antisense siNA cAAuuucucAucGAuuuccTT B 1589(3133C) stab19 3169 UCAUUGCCUAGAGGUGCUCAUUC 1284 36744 IL4R:3189L21antisense siNA AuGAGcAccucuAGGcAAuTT B 1590 (3171C) stab19 469CUAUACACUGGACCUGUGGGCUG 1277 36745 IL4R:489L21 antisense siNAGCCCACAGGUCCAGUGUAUTT B 1591 (471C) stab22 551 CCAGGAAACCUGACAGUUCACAC1278 36746 IL4R:571L21 antisense siNA GUGAACUGUCAGGUUUCCUTT B 1592(553C) stab22 1119 AGCACAACAUGAAAAGGGAUGAA 1279 36747 IL4R:1139L21antisense siNA CAUCCCUUUUCAUGUUGUGTTB 1593 (1121C) stab22 1120GCACAACAUGAAAAGGGAUGAAG 1280 36748 IL4R:1140L21 antisense siNAUCAUCCCUUUUCAUGUUGUTT B 1594 (1122C) stab22 1132 AAGGGAUGAAGAUCCUCACAAGG1281 36749 IL4R:1152L21 antisense siNA UUGUGAGGAUCUUCAUCCCTT B 1595(1134C) stab22 3130 UUGGGAAAUCGAUGAGAAAUUGA 1282 36750 IL4R:3150L21antisense siNA AAUUUCUCAUCGAUUUCCCTT B 1596 (3132C) stab22 3131UGGGAAAUCGAUGAGAAAUUGAA 1283 36751 IL4R:315IL21 antisense siNACAAUUUCUCAUCGAUUUCCTT B 1597 (3133C) stab22 3169 UCAUUGCCUAGAGGUGCUCAUUC1284 36752 IL4R:3189L21 antisense siNA AUGAGCACCUCUAGGCAAUTT B 1598(3171C) stab22 IL13 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:393U21 sensesiNA CAGUUUGUAAAGGACCUGCTT 1599 797 CACUUCACACACAGGCAACUGAG 1286IL13:799U21 sense siNA CUUCACACACAGGCAACUGTT 1600 832UCAGGCACACUUCUUCUUGGUCU 1287 IL13:834U21 sense siNAAGGCACACUUCUUCUUGGUTT 1601 911 AAGACUGUGGCUGCUAGCACUUG 1288 IL13:913U21sense siNA GACUGUGGCUGCUAGCACUTT 1602 963 AGCACUAAAGCAGUGGACACCAG 1289IL13:965U21 sense siNA CACUAAAGCAGUGGACACCTT 1603 965CACUAAAGCAGUGGACACCAGGA 1290 IL13:967U21 sense siNACUAAAGCAGUGGACACCAGTT 1604 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:970U21sense siNA AAGCAGUGGACACCAGGAGTT 1605 1191 AGAAGGGUACCUUGAACACUGGG 1292IL13:1193U21 sense siNA AAGGGUACCUUGAACACUGTT 1606 391CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNAGCAGGUCCUUUACAAACUGTT 1607 (393C) 797 CACUUCACACACAGGCAACUGAG 1286IL13:817L21 antisense siNA CAGUUGCCUGUGUGUGAAGTT 1608 (799C) 832UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNAACCAAGAAGAAGUGUGCCUTT 1609 (834C) 911 AAGACUGUGGCUGCUAGCACUUG 1288IL13:931L21 antisense siNA AGUGCUAGCAGCCACAGUCTT 1610 (913C) 963AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNAGGUGUCCACUGCUUUAGUGTT 1611 (965C) 965 CACUAAAGCAGUGGACACCAGGA 1290IL13:985L21 antisense siNA CUGGUGUCCACUGCUUUAGTT 1612 (967C) 968UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNACUCCUGGUGUCCACUGCUUTT 1613 (970C) 1191 AGAAGGGUACCUUGAACACUGGG 1292IL13:121IL21 antisense siNA CAGUGUUCAAGGUACCCUUTT 1614 (1193C) 391CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:393U21 sense siNA BcAGuuuGuAAAGGAccuGcTT B 1615 stab04 797 CACUUCACACACAGGCAACUGAG 1286IL13:799U21 sense siNA B cuucAcAcAcAGGcAAcuGTT B 1616 stab04 832UCAGGCACACUUCUUCUUGGUCU 1287 IL13:834U21 sense siNA BAGGcAcAcuucuucuuGGuTT B 1617 stab04 911 AAGACUGUGGCUGCUAGCACUUG 1288IL13:913U21 sense siNA B GAcuGuGGcuGcuAGcAcuTT B 1618 stab04 963AGCACUAAAGCAGUGGACACCAG 1289 IL13:965U21 sense siNA BcAcuAAAGcAGuGGAcAccTT B 1619 stab04 965 CACUAAAGCAGUGGACACCAGGA 1290IL13:967U21 sense siNA B cuAAAGcAGuGGAcAccAGTT B 1620 stab04 968UAAAGCAGUGGACACCAGGAGUC 1291 IL13:970U21 sense siNA BAAGcAGuGGAcAccAGGAGTT B 1621 stab04 1191 AGAAGGGUACCUUGAACACUGGG 1292IL13:1193U21 sense siNA B AAGGGuAccuuGAAcAcuGTT B 1622 stab04 391CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNAGcAGGuccuuuAcAAAcuGTsT 1623 (393C) stab05 797 CACUUCACACACAGGCAACUGAG1286 IL13:817L21 antisense siNA cAGuuGccuGuGuGuGAAGTsT 1624 (799C)stab05 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNAAccAAGAAGAAGuGuGccuTsT 1625 (834C) stab05 911 AAGACUGUGGCUGCUAGCACUUG1288 IL13:931L21 antisense siNA AGuGcuAGcAGccAcAGucTsT 1626 (913C)stab05 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNAGGuGuccAcuGcuuuAGuGTsT 1627 (965C) stab05 965 CACUAAAGCAGUGGACACCAGGA1290 IL13:985L21 antisense siNA cuGGuGuccAcuGcuuuAGTsT 1628 (967C)stab05 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNAcuccuGGuGuccAcuGcuuTsT 1629 (970C) stab05 1191 AGAAGGGUACCUUGAACACUGGG1292 IL13:121IL21 antisense siNA cAGuGuucAAGGuAcccuuTsT 1630 (1193C)stab05 864 UAUUGUGUGUUAUUUAAAUGAGU 1293 33355 IL13:864U21 sense siNA BuuGuGuGuuAuuuAAAuGATT B 1631 stab07 865 AUUGUGUGUUAUUUAAAUGAGUG 129433356 IL13:865U21 sense siNA B uGuGuGuuAuuuAAAuGAGTT B 1632 stab07 866UUGUGUGUUAUUUAAAUGAGUGU 1295 33357 IL13:866U21 sense siNA BGuGuGuuAuuuAAAuGAGuTT B 1633 stab07 863 UUAUUGUGUGUUAUUUAAAUGAG 129633358 IL13:863U21 sense siNA B AuuGuGuGuuAuuuAAAuGTT B 1634 stab07 200UGCAAUGGCAGCAUGGUAUGGAG 1297 33359 IL13:200U21 sense siNA BcAAuGGcAGcAuGGuAuGGTT B 1635 stab07 201 GCAAUGGCAGCAUGGUAUGGAGC 129833360 IL13:201U21 sense siNA B AAuGGcAGcAuGGuAuGGATT B 1636 stab07 202CAAUGGCAGCAUGGUAUGGAGCA 1299 33361 IL13:202U21 sense siNA BAuGGcAGcAuGGuAuGGAGTT B 1637 stab07 860 UUAUUAUUGUGUGUUAUUUAAAU 130033362 IL13:860U21 sense siNA B AuuAuuGuGuGuuAuuuAATT B 1638 stab07 861UAUUAUUGUGUGUUAUUUAAAUG 1301 33363 IL13:861U21 sense siNA BuuAuuGuGuGuuAuuuAAA TT B 1639 stab07 862 AUUAUUGUGUGUUAUUUAAAUGA 130233364 IL13:862U21 sense siNA B uAuuGuGuGuuAuuuAAAuTT B 1640 stab07 391CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:393U21 sense siNA BcAGuuuGuAAAGGAccuGcTT B 1641 stab07 797 CACUUCACACACAGGCAACUGAG 1286IL13:799U21 sense siNA B cuucAcAcAcAGGcAAcuGTT B 1642 stab07 832UCAGGCACACUUCUUCUUGGUCU 1287 IL13:834U21 sense siNA BAGGcAcAcuucuucuuGGuTT B 1643 stab07 911 AAGACUGUGGCUGCUAGCACUUG 1288IL13:913U21 sense siNA B GAcuGuGGcuGcuAGcAcuTT B 1644 stab07 963AGCACUAAAGCAGUGGACACCAG 1289 IL13:965U21 sense siNA BcAcuAAAGcAGuGGAcAccTT B 1645 stab07 965 CACUAAAGCAGUGGACACCAGGA 1290IL13:967U21 sense siNA B cuAAAGcAGuGGAcAccAGTT B 1646 stab07 968UAAAGCAGUGGACACCAGGAGUC 1291 IL13:970U21 sense siNA BAAGcAGuGGAcAccAGGAGTT B 1647 stab07 1191 AGAAGGGUACCUUGAACACUGGG 1292IL13:1193U21 sense siNA B AAGGGuAccuuGAAcAcuGTT B 1648 stab07 391CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNAGcAGGuccuuuAcAAAcuGTsT 1649 (393C) stab11 797 CACUUCACACACAGGCAACUGAG1286 IL13:817L21 antisense siNA cAGuuGccuGuGuGuGAAGTsT 1650 (799C)stab11 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNAAccAAGAAGAAGuGuGccuTsT 1651 (834C) stab11 911 AAGACUGUGGCUGCUAGCACUUG1288 IL13:931L21 antisense siNA AGuGcuAGcAGccAcAGucTsT 1652 (913C)stab11 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNAGGuGuccAcuGcuuuAGuGTsT 1653 (965C) stab11 965 CACUAAAGCAGUGGACACCAGGA1290 IL13:985L21 antisense siNA cuGGuGuccAcuGcuuuAGTsT 1654 (967C)stab11 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNAcuccuGGuGuccAcuGcuuTsT 1655 (970C) stab11 1191 AGAAGGGUACCUUGAACACUGGG1292 IL13:121IL21 antisense siNA cAGuGuucAAGGuAcccuuTsT 1656 (1193C)stab11 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:393U21 sense siNA BcAGuuuGuAAAGGAccuGcTT B 1657 stab18 797 CACUUCACACACAGGCAACUGAG 1286IL13:799U21 sense siNA B cuucAcAcAcAGGcAAcuGTT B 1658 stab18 832UCAGGCACACUUCUUCUUGGUCU 1287 IL13:834U21 sense siNA BAGGcAcAcuucuucuuGGuTT B 1659 stab18 911 AAGACUGUGGCUGCUAGCACUUG 1288IL13:913U21 sense siNA B GAcuGuGGcuGcuAGcAcuTT B 1660 stab18 963AGCACUAAAGCAGUGGACACCAG 1289 IL13:965U21 sense siNA BcAcuAAAGcAGuGGAcAccTT B 1661 stab18 965 CACUAAAGCAGUGGACACCAGGA 1290IL13:967U21 sense siNA B cuAAAGcAGuGGAcAccAGTT B 1662 stab18 968UAAAGCAGUGGACACCAGGAGUC 1291 IL13:970U21 sense siNA BAAGcAGuGGAcAccAGGAGTT B 1663 stab18 1191 AGAAGGGUACCUUGAACACUGGG 1292IL13:1193U21 sense siNA B AAGGGuAccuuGAAcAcuGTT B 1664 stab18 864UAUUGUGUGUUAUUUAAAUGAGU 1293 33375 IL13:882L21 antisense siNAucAuuuAAAuAAcAcAcAATsT 1665 (864C) stab08 865 AUUGUGUGUUAUUUAAAUGAGUG1294 33376 IL13:883L21 antisense siNA cucAuuuAAAuAAcAcAcATsT 1666 (865C)stab08 866 UUGUGUGUUAUUUAAAUGAGUGU 1295 33377 IL13:884L21 antisense siNAAcucAuuuAAAuAAcAcAcTsT 1667 (866C) stab08 863 UUAUUGUGUGUUAUUUAAAUGAG1296 33378 IL13:88IL21 antisense siNA cAuuuAAAuAAcAcAcAAuTsT 1668 (863C)stab08 200 UGCAAUGGCAGCAUGGUAUGGAG 1297 33379 IL13:218L21 antisense siNAccAuAccAuGcuGccAuuGTsT 1669 (200C) stab08 201 GCAAUGGCAGCAUGGUAUGGAGC1298 33380 IL13:219L21 antisense siNA uccAuAccAuGcuGccAuuTsT 1670 (201C)stab08 202 CAAUGGCAGCAUGGUAUGGAGCA 1299 33381 IL13:220L21 antisense siNAcuccAuAccAuGcuGccAuTsT 1671 (202C) stab08 860 UUAUUAUUGUGUGUUAUUUAAAU1300 33382 IL13:878L21 antisense siNA uuAAAuAAcAcAcAAuAAuTsT 1672 (860C)stab08 861 UAUUAUUGUGUGUUAUUUAAAUG 1301 33383 IL13:879L21 antisense siNAuuuAAAuAAcAcAcAAuAATsT 1673 (861C) stab08 862 AUUAUUGUGUGUUAUUUAAAUGA1302 33384 IL13:880L21 antisense siNA AuuuAAAuAAcAcAcAAuATsT 1674 (862C)stab08 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNAGcAGGuccuuuAcAAAcuGTsT 1675 (393C) stab08 797 CACUUCACACACAGGCAACUGAG1286 IL13:817L21 antisense siNA cAGuuGccuGuGuGuGAAGTsT 1676 (799C)stab08 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNAAccAAGAAGAAGuGuGccuTsT 1677 (834C) stab08 911 AAGACUGUGGCUGCUAGCACUUG1288 IL13:931L21 antisense siNA AGuGcuAGcAGccAcAGucTsT 1678 (913C)stab08 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNAGGuGuccAcuGcuuuAGuGTsT 1679 (965C) stab08 965 CACUAAAGCAGUGGACACCAGGA1290 IL13:985L21 antisense siNA cuGGuGuccAcuGcuuuAGTsT 1680 (967C)stab08 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNAcuccuGGuGuccAcuGcuuTsT 1681 (970C) stab08 1191 AGAAGGGUACCUUGAACACUGGG1292 IL13:121IL21 antisense siNA cAGuGuucAAGGuAcccuuTsT 1682 (1193C)stab08 391 CCCAGUUUGUAAAGGACCUGCUC 1285 36890 IL13:393U21 sense siNA BCAGUUUGUAAAGGACCUGCTT B 1683 stab09 797 CACUUCACACACAGGCAACUGAG 128636891 IL13:799U21 sense siNA B CUUCACACACAGGCAACUGTT B 1684 stab09 832UCAGGCACACUUCUUCUUGGUCU 1287 36892 IL13:834U21 sense siNA BAGGCACACUUCUUCUUGGUTT B 1685 stab09 911 AAGACUGUGGCUGCUAGCACUUG 128836893 IL13:913U21 sense siNA B GACUGUGGCUGCUAGCACUTT B 1686 stab09 963AGCACUAAAGCAGUGGACACCAG 1289 36894 IL13:965U21 sense siNA BCACUAAAGCAGUGGACACCTT B 1687 stab09 965 CACUAAAGCAGUGGACACCAGGA 129036895 IL13:967U21 sense siNA B CUAAAGCAGUGGACACCAGTT B 1688 stab09 968UAAAGCAGUGGACACCAGGAGUC 1291 36896 IL13:970U21 sense siNA BAAGCAGUGGACACCAGGAGTT B 1689 stab09 1191 AGAAGGGUACCUUGAACACUGGG 129236897 IL13:1193U21 sense siNA B AAGGGUACCUUGAACACUGTT B 1690 stab09 391CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNAGCAGGUCCUUUACAAACUGTsT 1691 (393C) stab10 797 CACUUCACACACAGGCAACUGAG1286 IL13:817L21 antisense siNA CAGUUGCCUGUGUGUGAAGTsT 1692 (799C)stab10 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNAACCAAGAAGAAGUGUGCCUTsT 1693 (834C) stab10 911 AAGACUGUGGCUGCUAGCACUUG1288 IL13:931L21 antisense siNA AGUGCUAGCAGCCACAGUCTsT 1694 (913C)stab10 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNAGGUGUCCACUGCUUUAGUGTsT 1695 (965C) stab10 965 CACUAAAGCAGUGGACACCAGGA1290 IL13:985L21 antisense siNA CUGGUGUCCACUGCUUUAGTsT 1696 (967C)stab10 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNACUCCUGGUGUCCACUGCUUTsT 1697 (970C) stab10 1191 AGAAGGGUACCUUGAACACUGGG1292 IL13:121IL21 antisense siNA CCAGUGUUCAAGGUACCCUUTsT 1698 (1193C)stab10 391 CCCAGUUUGUAAAGGACCUGCUC 1285 IL13:411L21 antisense siNAGcAGGuccuuuAcAAAcuGTT B 1699 (393C) stab19 797 CACUUCACACACAGGCAACUGAG1286 IL13:817L21 antisense siNA cAGuuGccuGuGuGuGAAGTT B 1700 (799C)stab19 832 UCAGGCACACUUCUUCUUGGUCU 1287 IL13:852L21 antisense siNAAccAAGAAGAAGuGuGccuTT B 1701 (834C) stab19 911 AAGACUGUGGCUGCUAGCACUUG1288 IL13:931L21 antisense siNA AGuGcuAGcAGccAcAGucTT B 1702 (913C)stab19 963 AGCACUAAAGCAGUGGACACCAG 1289 IL13:983L21 antisense siNAGGuGuccAcuGcuuuAGuGTT B 1703 (965C) stab19 965 CACUAAAGCAGUGGACACCAGGA1290 IL13:985L21 antisense siNA cuGGuGuccAcuGcuuuAGTT B 1704 (967C)stab19 968 UAAAGCAGUGGACACCAGGAGUC 1291 IL13:988L21 antisense siNAcuccuGGuGuccAcuGcuuTT B 1705 (970C) stab19 1191 AGAAGGGUACCUUGAACACUGGG1292 IL13:121IL21 antisense siNA cAGuGuucAAGGuAcccuuTT B 1706 (1193C)stab19 391 CCCAGUUUGUAAAGGACCUGCUC 1285 36898 IL13:411L21 antisense siNAGCAGGUCCUUUACAAACUGTT B 1707 (393C) stab22 797 CACUUCACACACAGGCAACUGAG1286 36899 IL13:817L21 antisense siNA CAGUUGCCUGUGUGUGAAGTT B 1708(799C) stab22 832 UCAGGCACACUUCUUCUUGGUCU 1287 36900 IL13:852L21antisense siNA ACCAAGAAGAAGUGUGCCUTT B 1709 (834C) stab22 911AAGACUGUGGCUGCUAGCACUUG 1288 36901 IL13:931L21 antisense siNAAGUGCUAGCAGCCACAGUCTTB 1710 (913C) stab22 963 AGCACUAAAGCAGUGGACACCAG1289 36902 IL13:983L21 antisense siNA GGUGUCCACUGCUUUAGUGTT B 1711(965C) stab22 965 CACUAAAGCAGUGGACACCAGGA 1290 36903 IL13:985L21antisense siNA CUGGUGUCCACUGCUUUAGTT B 1712 (967C) stab22 968UAAAGCAGUGGACACCAGGAGUC 1291 36904 IL13:988L21 antisense siNACUCCUGGUGUCCACUGCUUTT B 1713 (970C) stab22 1191 AGAAGGGUACCUUGAACACUGGG1292 36905 IL13:121IL21 antisense siNA CAGUGUUCAAGGUACCCUUTT B 1714(1193C) stab22 IL13R 408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:410U21sense siNA GGUGAUCCUGAGUCUGCUGTT 1715 657 UGGUCAAGGAUAAUGCAGGAAAA 1304IL13RA1:659U21 sense siNA GUCAAGGAUAAUGCAGGAATT 1716 871CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:873U21 sense siNAUCCAAGAGGCUAAAUGUGATT 1717 1276 GGAAACCGACUCUGUAGUGCUGA 1306IL13RA1:1278U21 sense siNA AAACCGACUCUGUAGUGCUTT 1718 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1310U21 sense siNAAAGAAAGCCUCUCAGUGAUTT 1719 1424 ACUGCACCAUUUAAAAACAGGCA 1308IL13RA1:1426U21 sense siNA UGCACCAUUUAAAAACAGGTT 1720 2186CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2188U21 sense siNAGCAUUUUCCUCUGCUUUGATT 1721 2270 CCAAGACCUUUCAAAGCCAUUUU 1310IL13RA1:2272U21 sense siNA AAGACCUUUCAAAGCCAUUTT 1722 408AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisenseCAGCAGACUCAGGAUCACCTT 1723 siNA (410C) 657 UGGUCAAGGAUAAUGCAGGAAAA 1304IL13RA1:677L21 antisense UUCCUGCAUUAUCCUUGACTT 1724 siNA (659C) 871CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:89IL21 antisenseUCACAUUUAGCCUCUUGGATT 1725 siNA (873C) 1276 GGAAACCGACUCUGUAGUGCUGA 1306IL13RA1:1296L21 antisense AGCACUACAGAGUCGGUUUTT 1726 siNA (1278C) 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisenseAUCACUGAGAGGCUUUCUUTT 1727 siNA (1310C) 1424 ACUGCACCAUUUAAAAACAGGCA1308 IL13RA1:1444L21 antisense CCUGUUUUUAAAUGGUGCATT 1728 siNA (1426C)2186 CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisenseUCAAAGCAGAGGAAAAUGCTT 1729 siNA (2188C) 2270 CCAAGACCUUUCAAAGCCAUUUU1310 IL13RA1:2290L21 antisense AAUGGCUUUGAAAGGUCUUTT 1730 siNA (2272C)408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:410U21 sense siNA BGGuGAuccuGAGucuGcuGTT B 1731 stab04 657 UGGUCAAGGAUAAUGCAGGAAAA 1304IL13RA1:659U21 sense siNA B GucAAGGAuAAuGcAGGAATT B 1732 stab04 871CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:873U21 sense siNA BuccAAGAGGcuAAAuGuGATT B 1733 stab04 1276 GGAAACCGACUCUGUAGUGCUGA 1306IL13RA1:1278U21 sense siNA B AAAccGAcucuGuAGuGcuTT B 1734 stab04 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1310U21 sense siNA BAAGAAAGccucucAGuGAuTT B 1735 stab04 1424 ACUGCACCAUUUAAAAACAGGCA 1308IL13RA1:1426U21 sense siNA B uGcAccAuuuAAAAAcAGGTT B 1736 stab04 2186CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2188U21 sense siNA BGcAuuuuccucuGcuuuGATT B 1737 stab04 2270 CCAAGACCUUUCAAAGCCAUUUU 1310IL13RA1:2272U21 sense siNA B AAGAccuuucAAAGccAuuTT B 1738 stab04 408AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisensecAGcAGAcucAGGAucAccTsT 1739 siNA (410C) stab05 657UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisenseuuccuGcAuuAuccuuGAcTsT 1740 siNA (659C) stab05 871CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:891L21 antisenseucAcAuuuAGccucuuGGATsT 1741 siNA (873C) stab05 1276GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisenseAGcAcuAcAGAGucGGuuuTsT 1742 siNA (1278C) stab05 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisenseAucAcuGAGAGGcuuucuuTsT 1743 siNA (1310C) stab05 1424ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisenseccuGuuuuuAAAuGGuGcATsT 1744 siNA (1426C) stab05 2186CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisenseucAAAGcAGAGGAAAAuGcTsT 1745 siNA (2188C) stab05 2270CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisenseAAuGGcuuuGAAAGGucuuTsT 1746 siNA (2272C) stab05 408AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:410U21 sense siNA BGGuGAuccuGAGucuGcuGTT B 1747 stab07 657 UGGUCAAGGAUAAUGCAGGAAAA 1304IL13RA1:659U21 sense siNA B GucAAGGAuAAuGcAGGAATT B 1748 stab07 871CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:873U21 sense siNA BuccAAGAGGcuAAAuGuGATT B 1749 stab07 1276 GGAAACCGACUCUGUAGUGCUGA 1306IL13RA1:1278U21 sense siNA B AAAccGAcucuGuAGuGcuTT B 1750 stab07 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1310U21 sense siNA BAAGAAAGccucucAGuGAuTT B 1751 stab07 1424 ACUGCACCAUUUAAAAACAGGCA 1308IL13RA1:1426U21 sense siNA B uGcAccAuuuAAAAAcAGGTT B 1752 stab07 2186CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2188U21 sense siNA BGcAuuuuccucuGcuuuGATT B 1753 stab07 2270 CCAAGACCUUUCAAAGCCAUUUU 1310IL13RA1:2272U21 sense siNA B AAGAccuuucAAAGccAuuTT B 1754 stab07 408AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisensecAGcAGAcucAGGAucAccTsT 1755 siNA (410C) stab11 657UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisenseuuccuGcAuuAuccuuGAcTsT 1756 siNA (659C) stab11 871CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:891L21 antisenseucAcAuuuAGccucuuGGATsT 1757 siNA (873C) stab11 1276GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisenseAGcAcuAcAGAGucGGuuuTsT 1758 siNA (1278C) stab11 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisenseAucAcuGAGAGGcuuucuuTsT 1759 siNA (1310C) stab11 1424ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisenseccuGuuuuuAAAuGGuGcATsT 1760 siNA (1426C) stab11 2186CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisenseucAAAGcAGAGGAAAAuGcTsT 1761 siNA (2188C) stab11 2270CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisenseAAuGGcuuuGAAAGGucuuTsT 1762 siNA (2272C) stab11 408AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:410U21 sense siNA BGGuGAuccuGAGucuGcuGTT B 1763 stab18 657 UGGUCAAGGAUAAUGCAGGAAAA 1304IL13RA1:659U21 sense siNA B GucAAGGAuAAuGcAGGAATT B 1764 stab18 871CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:873U21 sense siNA BuccAAGAGGcuAAAuGuGATT B 1765 stab18 1276 GGAAACCGACUCUGUAGUGCUGA 1306IL13RA1:1278U21 sense siNA B AAAccGAcucuGuAGuGcuTT B 1766 stab18 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1310U21 sense siNA BAAGAAAGccucucAGuGAuTT B 1767 stab18 1424 ACUGCACCAUUUAAAAACAGGCA 1308ILl3RAl:1426U21 sense siNA B uGcAccAuuuAAAAAcAGGTT B 1768 stab18 2186CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2188U21 sense siNA BGcAuuuuccucuGcuuuGATT B 1769 stab18 2270 CCAAGACCUUUCAAAGCCAUUUU 1310IL13RA1:2272U21 sense siNA B AAGAccuuucAAAGccAuuTT B 1770 stab18 408AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisensecAGcAGAcucAGGAucAccTsT 1771 siNA (410C) stab08 657UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisenseuuccuGcAuuAuccuuGAcTsT 1772 siNA (659C) stab08 8710GU00AAGAGG0UAAAUGUGAGA 1305 L13RA1:891L21 antisenseucAcAuuuAGccucuuGGATsT 1773 siNA (873C) stab08 1276GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisenseAGcAcuAcAGAGucGGuuuTsT 1774 siNA (1278C) stab08 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisenseAucAcuGAGAGGcuuucuuTsT 1775 siNA (1310C) stab08 1424ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisenseccuGuuuuuAAAuGGuGcATsT 1776 siNA (1426C) stab08 2186CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisenseucAAAGcAGAGGAAAAuGcTsT 1777 siNA (2188C) stab08 2270CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisenseAAuGGcuuuGAAAGGucuuTsT 1778 siNA (2272C) stab08 408AAGGUGAUCCUGAGUCUGCUGUG 1303 36906 IL13RA1:410U21 sense siNA BGGUGAUCCUGAGUCUGCUGTT B 1779 stab09 657 UGGUCAAGGAUAAUGCAGGAAAA 130436907 IL13RA1:659U21 sense siNA B GUCAAGGAUAAUGCAGGAATT B 1780 stab09871 CGUCCAAGAGGCUAAAUGUGAGA 1305 36908 IL13RA1:873U21 sense siNA BUCCAAGAGGCUAAAUGUGATT B 1781 stab09 1276 GGAAACCGACUCUGUAGUGCUGA 130636909 IL13RA1:1278U21 sense siNA B AAACCGACUCUGUAGUGCUTT B 1782 stab091308 UGAAGAAAGCCUCUCAGUGAUGG 1307 36910 IL13RA1:1310U21 sense siNA BAAGAAAGCCUCUCAGUGAUTT B 1783 stab09 1424 ACUGCACCAUUUAAAAACAGGCA 130836911 IL13RA1:1426U21 sense siNA B UGCACCAUUUAAAAACAGGTT B 1784 stab092186 CAGCAUUUUCCUCUGCUUUGAAA 1309 36912 IL13RA1:2188U21 sense siNA BGCAUUUUCCUCUGCUUUGATT B 1785 stab09 2270 CCAAGACCUUUCAAAGCCAUUUU 131036913 IL13RA1:2272U21 sense siNA B AAGACCUUUCAAAGCCAUUTT B 1786 stab09408 AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisenseCAGCAGACUCAGGAUCACCTsT 1787 siNA (410C) stab10 657UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisenseUUCCUGCAUUAUCCUUGACTsT 1788 siNA (659C) stab10 871CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:891L21 antisenseUCACAUUUAGCCUCUUGGATsT 1789 siNA (873C) stab10 1276GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisenseAGCACUACAGAGUCGGUUUTsT 1790 siNA (1278C) stab10 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisenseAUCACUGAGAGGCUUUCUUTsT 1791 siNA (1310C) stab10 1424ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisenseCCUGUUUUUAAAUGGUGCATsT 1792 siNA (1426C) stab10 2186CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisenseUCAAAGCAGAGGAAAAUGCTsT 1793 siNA (2188C) stab10 2270CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisenseAAUGGCUUUGAAAGGUCUUTsT 1794 siNA (2272C) stab10 408AAGGUGAUCCUGAGUCUGCUGUG 1303 IL13RA1:428L21 antisensecAGcAGAcucAGGAucAccTT B 1795 siNA (410C) stab19 657UGGUCAAGGAUAAUGCAGGAAAA 1304 IL13RA1:677L21 antisenseuuccuGcAuuAuccuuGAcTT B 1796 siNA (659C) stab19 871CGUCCAAGAGGCUAAAUGUGAGA 1305 IL13RA1:891L21 antisenseucAcAuuuAGccucuuGGATT B 1797 siNA (873C) stab19 1276GGAAACCGACUCUGUAGUGCUGA 1306 IL13RA1:1296L21 antisenseAGcAcuAcAGAGucGGuuuTT B 1798 siNA (1278C) stab19 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 IL13RA1:1328L21 antisenseAucAcuGAGAGGcuuucuuTT B 1799 siNA (1310C) stab19 1424ACUGCACCAUUUAAAAACAGGCA 1308 IL13RA1:1444L21 antisenseccuGuuuuuAAAuGGuGcATT B 1800 siNA (1426C) stab19 2186CAGCAUUUUCCUCUGCUUUGAAA 1309 IL13RA1:2206L21 antisenseucAAAGcAGAGGAAAAuGcTT B 1801 siNA (2188C) stab19 2270CCAAGACCUUUCAAAGCCAUUUU 1310 IL13RA1:2290L21 antisenseAAuGGcuuuGAAAGGucuuTT B 1802 siNA (2272C) stab19 408AAGGUGAUCCUGAGUCUGCUGUG 1303 36914 IL13RA1:428L21 antisenseCAGCAGACUCAGGAUCACCTT B 1803 siNA (410C) stab22 657UGGUCAAGGAUAAUGCAGGAAAA 1304 36915 IL13RA1:677L21 antisenseUUCCUGCAUUAUCCUUGACTT B 1804 siNA (659C) stab22 871CGUCCAAGAGGCUAAAUGUGAGA 1305 36916 IL13RA1:891L21 antisenseUCACAUUUAGCCUCUUGGATT B 1805 siNA (873C) stab22 1276GGAAACCGACUCUGUAGUGCUGA 1306 36917 IL13RA1:1296L21 antisenseAGCACUACAGAGUCGGUUUTT B 1806 siNA (1278C) stab22 1308UGAAGAAAGCCUCUCAGUGAUGG 1307 36918 IL13RA1:1328L21 antisenseAUCACUGAGAGGCUUUCUUTT B 1807 siNA (1310C) stab22 1424ACUGCACCAUUUAAAAACAGGCA 1308 36919 IL13RA1:1444L21 antisenseCCUGUUUUUAAAUGGUGCATT B 1808 siNA (1426C) stab22 2186CAGCAUUUUCCUCUGCUUUGAAA 1309 36920 IL13RA1:2206L21 antisenseUCAAAGCAGAGGAAAAUGCTT B 1809 siNA (2188C) stab22 2270CCAAGACCUUUCAAAGCCAUUUU 1310 36921 IL13RA1:2290L21 antisenseAAUGGCUUUGAAAGGUCUUTT B 1810 siNA (2272C) stab22 Uppercase= ribonucleotide u, c = 2′-deoxy-2′-fluoro U, C T = thymidine B= inverted deoxy abasic s = phosphorothioate linkage A = deoxy AdenosineG = deoxy Guanosine G = 2′-O-methyl Guanosine A = 2′-O-methyl Adenosine

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at S/AS 3′-ends “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All Usually AS linkages“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4”2′-fluoro Ribo 5′ and — Usually S 3′-ends “Stab 5” 2′-fluoro Ribo — 1 at3′-end Usually AS “Stab 6” 2′-O-Methyl Ribo 5′ and — Usually S 3′-ends“Stab 7” 2′-fluoro 2′-deoxy 5′ and — Usually S 3′-ends “Stab 8”2′-fluoro 2′-O- — 1 at 3′-end Usually AS Methyl “Stab 9” Ribo Ribo 5′and — Usually S 3′-ends “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS“Stab 11” 2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12”2′-fluoro LNA 5′ and Usually S 3′-ends “Stab 13” 2′-fluoro LNA 1 at3′-end Usually AS “Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1at 3′-end “Stab 15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end“Stab 16” Ribo 2′-O- 5′ and Usually S Methyl 3′-ends “Stab 17”2′-O-Methyl 2′-O- 5′ and Usually S Methyl 3′-ends “Stab 18” 2′-fluoro2′-O- 5′ and 1 at 3′-end Usually S Methyl 3′-ends “Stab 19” 2′-fluoro2′-O- 3′-end Usually AS Methyl “Stab 20” 2′-fluoro 2′-deoxy 3′-endUsually AS “Stab 21” 2′-fluoro Ribo 3′-end Usually AS “Stab 22” RiboRibo 3′-end - Usually AS “Stab 23” 2′-fluoro* 2′-deoxy* 5′ and Usually S3′-ends “Stab 24” 2′-fluoro* 2′-O- — 1 at 3′-end Usually AS Methyl*“Stab 25” 2′-fluoro* 2′-O- — 1 at 3′-end Usually AS Methyl* CAP = anyterminal cap, see for example FIG. 10. All Stab 1-25 chemistries cancomprise 3′-terminal thymidine (TT) residues All Stab 1-25 chemistriestypically comprise about 21 nucleotides, but can vary as describedherein. S = sense strand AS = antisense strand *Stab 23 has singleribonucleotide adjacent to 3′-CAP *Stab 24 has single ribonucleotide at5′-terminus *Stab 25 has three ribonucleotides at 5′-terminus

TABLE V Reagent Equivalents Amount Wait Time* DNA Wait Time* 2′-O-methylWait Time* RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124 μL 5 sec5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 secAcetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 wellInstrument Equivalents: DNA/ Amount: DNA/2′-O- Wait Time* 2′-O- Reagent2′-O-methyl/Ribo methyl/Ribo Wait Time* DNA methyl Wait Time* RiboPhosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-EthylTetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec  200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

1. A chemically modified nucleic acid molecule, wherein: (a) the nucleicacid molecule comprises a sense strand having SEQ ID NO:1503 and aseparate antisense strand having SEQ ID NO:1511; (b) 50 percent or moreof the nucleotides in each strand comprise a 2′-sugar modification,wherein the 2′-sugar modification of any of the pyrimidine nucleotidesdiffers from the 2′-sugar modification of any of the purine nucleotides.2. The nucleic acid molecule of claim 1, wherein the 2′-sugarmodification of any of the purine nucleotides in the sense stranddiffers from the 2′-sugar modification of any of the purine nucleotidesin the antisense strand.
 3. The nucleic acid molecule of claim 1,wherein the 2′-sugar modification is selected from the group consistingof 2′-deoxy-2′-fluoro, 2′-O-methyl, and 2′-deoxy.
 4. The nucleic acid ofclaim 3, wherein the 2′-deoxy-2′-fluoro sugar modification is apyrimidine modification.
 5. The nucleic acid of claim 3, wherein the2′-deoxy sugar modification is a pyrimidine modification.
 6. The nucleicacid of claim 3, wherein the 2′-O-methyl sugar modification is apyrimidine modification.
 7. The nucleic acid molecule of claim 4,wherein said pyrimidine modification is in the sense strand, theantisense strand, or both the sense strand and antisense strand.
 8. Thenucleic acid molecule of claim 6, wherein said pyrimidine modificationis in the sense strand, the antisense strand, or both the sense strandand antisense strand.
 9. The nucleic acid molecule of claim 3, whereinthe 2 ′-deoxy sugar modification is a purine modification.
 10. Thenucleic acid molecule of claim 3, wherein the 2′-O-methyl sugarmodification is a purine modification.
 11. The nucleic acid molecule ofclaim 9, wherein the purine modification is in the sense strand.
 12. Thenucleic acid molecule of claim 10, wherein the purine modification is inthe antisense strand.
 13. The nucleic acid molecule of claim 1, whereinthe nucleic acid molecule comprises ribonucleotides.
 14. The nucleicacid molecule of claim 1, wherein the sense strand includes a terminalcap moiety at the 5′-end, the 3′-end, or both of the 5′- and 3′- ends.15. The nucleic acid molecule of claim 14, wherein the terminal capmoiety is an inverted deoxy abasic moiety.
 16. The nucleic acid moleculeof claim 1, wherein said nucleic acid molecule includes one or morephosphorothioate internucleotide linkages.
 17. The nucleic acid moleculeof claim 16, wherein one of the phosphorothioate internucleotidelinkages is at the 3′-end of the antisense strand.
 18. The nucleic acidmolecule of claim 1, wherein the 5′-end of the antisense strand includesa terminal phosphate group.
 19. A composition comprising the nucleicacid molecule of claim 1, in a pharmaceutically acceptable carrier ordiluent.